JP3557277B2 - Thermostable trehalose releasing enzyme, its production method and use - Google Patents

Abstract

Disclosed is a thermostable trehalose-releasing enzyme, and its preparations and uses. The enzyme is obtainable from the culture of microorganisms such as Sulfolobus acidocaldarius (ATCC 33909 and ATCC 49426) and Sulfolobus solfataricus (ATCC 35091 and ATCC 35092), and capable of hydrolyzing at a temperature of over 55 DEG C the linkage between a trehalose moiety and the remaining glycosyl moiety in a non-reducing saccharide having a trehalose structure as an end unit and having a degree of glucose polymerization of 3 or higher. Trehalose and compositions containing the same are extensively useful in food products, cosmetics and pharmaceuticals. <IMAGE>

Description

【００４８】
【表２】

【００４９】
表１及び表２の工程でそれぞれゲル瀘過溶出液として得られた、精製耐熱性非還元性糖質生成酵素及び精製耐熱性トレハロース遊離酵素をポリアクリルアミドゲル（ゲル濃度７．５％）を用いる電気泳動法で純度を検定したところ、蛋白バンドは単一で、純度の高い標品であった。
【００５０】
【実験３ 理化学的性質】
【実験３−１ 耐熱性トレハロース遊離酵素の性質】
実験２の方法で得られた精製耐熱性トレハロース遊離酵素をＳＤＳ−ポリアクリルアミドゲル（ゲル濃度１０％）を用いる電気泳動法に供し、同時に泳動した分子量マーカー（日本バイオ・ラッド・ラボラトリーズ株式会社製）と比較して本酵素の分子量を測定したところ、分子量約５４，０００乃至６４，０００ダルトンであった。
【００５１】
本精製酵素をポリアクリルアミドゲル（２％アンフォライン含有、ファルマシア・エルケービー社製）を用いる等電点電気泳動法に供し、泳動後、ゲルのｐＨを測定して本酵素の等電点を求めたところ、等電点は約５．６乃至６．６であった。
【００５２】
本酵素活性に対するの温度の影響、ｐＨの影響を活性測定方法に準じて調べた。結果を図２（温度の影響）、図３（ｐＨの影響）に示した。酵素の至適温度はｐＨ６．０、３０分間反応で約７５℃、至適ｐＨは６０℃、３０分間反応で約５．５乃至６．０であった。本酵素の温度安定性は、酵素溶液（５０ｍＭリン酸緩衝液を含む、ｐＨ７．０）を各温度に６０分間保持し、水冷した後、残存する酵素活性を測定することにより求めた。また、ｐＨ安定性は、本酵素を各ｐＨの５０ｍＭ緩衝液中で２５℃、１６時間保持した後、ｐＨを７に調整し、残存する酵素活性を測定することにより求めた。それぞれの結果を図４（温度安定性）、図５（ｐＨ安定性）に示した。本酵素の温度安定性は約８５℃までであり、ｐＨ安定性は５．５乃至９．５であった。
【００５３】
本精製酵素のＮ末端アミノ酸配列を、アプライド・バイオシステムズ・ジャパン販売プロテインシーケンサー モデル『４７３Ａ』を用いて、Ｎ末端から１０残基まで分析したところ、本酵素のＮ末端アミノ酸配列は、メチオニン−フェニルアラニン−セリン−フェニルアラニン−グリシン−グリシン−アスパラギン−イソロイシン−グルタミン酸−リジンであった。
【００５４】
【実験３−２ 耐熱性非還元性糖質生成酵素の理化学的性質】
実験２の方法で得られた精製耐熱性非還元性糖質生成酵素をＳＤＳ−ポリアクリルアミドゲル（ゲル濃度１０％）を用いる電気泳動法に供し、同時に泳動した分子量マーカーと比較して本酵素の分子量を測定したところ、分子量約６９，０００乃至７９，０００ダルトンであった。本精製酵素をポリアクリルアミドゲルを用いる等電点電気泳動法に供し、泳動後、ゲルのｐＨを測定して本酵素の等電点を求めたところ、等電点は約５．４乃至６．４であった。
【００５５】
本酵素活性に対するの温度の影響、ｐＨの影響を活性測定方法に準じて調べた。酵素の至適温度は約７５℃、至適ｐＨは約５．０乃至５．５であった。本酵素の温度安定性及びｐＨ安定性を実験３−１の方法と同様に調べたところ、本酵素の温度安定性は約８５℃までであり、ｐＨ安定性は４．０乃至９．５であった。
【００５６】
本精製酵素のＮ末端アミノ酸配列を実験３−１の方法と同様に調べたところ、本酵素のＮ末端アミノ酸配列は、メチオニン−イソロイシン−セリン−アラニン−スレオニン−チロシン−アルギニン−ロイシン−グルタミン−ロイシンであった。
【００５７】
【実験４ 耐熱性トレハロース遊離酵素によるトレハロースの生成】
【実験４−１ 非還元性糖質生成酵素の調製】
５００ｍｌ容三角フラスコにマルトース２．０％（ｗ／ｖ）、ペプトン０．５％（ｗ／ｖ）、酵母エキス０．１％（ｗ／ｖ）、リン酸水素二ナトリウム０．１％及びリン酸二水素カリウム０．１％を含む液体培地（ｐＨ７．０）を１００ｍｌずつとり、１２０℃で２０分間オートクレーブして滅菌した。冷却後、三角フラスコ内の液体培地にリゾビウム・スピーシーズＭ−１１（ＦＥＲＭ ＢＰ−４１３０）を植菌し、回転振盪下、２７℃で２４時間種培養した。別途、３０ｌ容ファーメンターに同組成の液体培地を２０ｌとり、滅菌後、上記で得た種培養液を１ｖ／ｖ％接種し、液体培地をｐＨ６乃至８に保ちつつ、３０℃で７２時間通気撹拌培養した。培養物を大日本製薬株式会社製超高圧菌体破砕装置『ミニラボ』で処理し、含まれる菌体を破砕した。遠心分離により不溶物を除去後、硫安分画、ＤＥＡＥ−トヨパールを用いたイオン交換カラムクロマトグラフィー、ブチルトヨパールを用いた疎水カラムクロマトグラフィー、トヨパールＨＷ−５５を用いたゲル瀘過カラムクロマトグラフィーを行って精製したところ、比活性１９５単位／ｍｇ蛋白質の非還元性糖質生成酵素が、培養１ｌ当たりに換算して、約２２０単位の収量で得られた。
【００５８】
なお、リゾビウム・スピーシーズＭ−１１由来非還元性糖質生成酵素の活性測定は、実験２の測定条件のうち、５０ｍＭ酢酸緩衝液（ｐＨ５．５）を５０ｍＭリン酸緩衝液（ｐＨ７．０）に、反応温度を６０℃を４０℃に代えて行った。
【００５９】
【実験４−２ 末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質の調製】
マルトトリオース、マルトテトラオース、マルトペンタオース、マルトヘキサオース及びマルトヘプタオースから選ばれる還元性澱粉部分分解物の２０％水溶液に実験４−１の方法で得られた非還元性糖質生成酵素を基質固形物グラム当たりそれぞれ２単位の割合で加え、４０℃、ｐＨ７．０で４８時間作用させた後、常法に従って、加熱失活、瀘過、脱色、脱塩、濃縮し、東京有機化学工業株式会社製ナトリウム型強酸性カチオン交換樹脂『ＸＴ−１０１６』（架橋度４％）を用いたイオン交換カラムクロマトグラフィーを行った。樹脂を内径２．０ｃｍ、長さ１ｍのジャッケト付ステンレス製カラム３本に充填し、直列につなぎ、カラム内温度を５５℃に維持しつつ、反応糖液を樹脂に対して５ｖ／ｖ％加え、これに５５℃の温水をＳＶ０．１３で流して分画し、末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質の純度９５％以上の画分を回収した。回収した画分に水酸化ナトリウムを０．１Ｎになるように加え、１００℃で２時間加熱して残存する還元性糖質を分解した。この溶液を活性炭にて脱色し、Ｈ型、ＯＨ型、イオン交換樹脂で脱塩し、純度９９．０％以上のα−グルコシルトレハロース、α−マルトシルトレハロース、α−マルトトリオシルトレハロース、α−マルトテトラオシルトレハロース、α−マルトペンタオシルトレハロースの非還元性糖質標品を調製した。
【００６０】
【実験４−３ 耐熱性トレハロース遊離酵素による非還元性糖質からのトレハロースの生成】
実験４−２の方法で得られた５種の非還元性糖質の５％水溶液を調製し、それぞれに実験２で得られた耐熱性トレハロース遊離酵素を基質固形物グラム当たり２単位の割合で加え、６０℃、ｐＨ５．５で４８時間作用させた後、脱塩し、和光純薬工業株式会社製カラム『ワコービーズ ＷＢ−Ｔ−３３０』を用いた高速液体クロマトグラフィーで反応生成物を分析した。対照として、マルトトリオース、マルトテトラオース、マルトペンタオース、マルトヘキサオース、マルトヘプタオースに耐熱性トレハロース遊離酵素を同様に作用させ、高速液体クロマトグラフィーで分析した。それらの結果を表３示す。
【００６１】
【表３】

【００６２】
表３の結果から明らかなように、
（１） 耐熱性トレハロース遊離酵素は、末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質のトレハロース部分とグリコシル部分との間の結合を特異的に加水分解し、トレハロースとグルコース重合度が１以上の還元性糖質とを生成する。（２） マルトオリゴ糖は、耐熱性トレハロース遊離酵素によって全く作用をうけない。
【００６３】
これらの結果から、本発明の耐熱性トレハロース遊離酵素は、末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質のトレハロース部分とその他のグリコシル部分との間の結合を極めて特異的に加水分解し、トレハロースを遊離する全く新しい作用機構の酵素であると判断される。
【００６４】
【実験５ 還元性澱粉部分分解物からのトレハロースの調製】
５％ワキシーコーンスターチ懸濁液を加熱糊化させた後、ｐＨ４．５、温度５０℃に調整し、これにイソアミラーゼ（株式会社林原生物化学研究所製）を澱粉グラム当たり４０００単位の割合になるように加え、２０時間反応させた。その反応液をオートクレーブ（１２０℃、１０分間）し、次いで６０℃に冷却し、これを東ソー株式会社製カラム『トヨパールカラムＨＷ５０』を用いたゲル瀘過クロマトグラフィー（ゲル量７５０ｍｌ）でグルコース重合度３７乃至１１の還元性澱粉部分分解物を分離した。
【００６５】
得られた還元性澱粉部分分解物、又はグルコース重合度３のマルトトリオースを、１０ｍＭリン酸緩衝液（ｐＨ５．５）で１％濃度に調整し、これに実験２の方法で調製した精製耐熱性非還元性糖質生成酵素標品及び精製耐熱性トレハロース遊離酵素標品をそれぞれ基質固形物当たり４単位の割合で加え、６０℃で２４時間作用させた後、一部を採り、脱塩し、高速液体クロマトグラフィーで反応生成物を分析した。残りの反応液は、更に、５０℃、ｐＨ４．５に調整した後、グルコアミラーゼ（生化学工業株式会社製）を基質固形物当たり５０単位の割合で加え、１０時間作用させ、同様に脱塩し、高速液体クロマトグラフィーで反応生成物を分析した。それらの結果を表４に示す。
【００６６】
【表４】

【００６７】
表４に示すように、耐熱性非還元性糖質生成酵素及び耐熱性トレハロース遊離酵素を作用させた後のトレハロース生成率は、グルコース重合度３のマルトトリオースでは２．２％と低い値であったが、グルコース重合度１０．８乃至３６．８の澱粉部分分解物では６３．３乃至８１．２％の高い値が得られた。また、グルコース重合度が高い程、得られるトレハロース純度が高いことも判明した。更に、グルコアミラーゼで残存する末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質をトレハロースとグルコースとに分解することにより、生成するトレハロース純度がより高まることも判明した。
【００６８】
【実験６ 他のスルフォロブス属微生物由来の耐熱性トレハロース遊離酵素の生産とその性質】
スルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９に代えて、スルフォロブス・アシドカルダリウス ＡＴＣＣ４９４２６、スルフォロブス・ソルファタリカス ＡＴＣＣ３５０９１、スルフォロブス・ソルファタリカス ＡＴＣＣ３５０９２を用いた以外は、実験１と同様にファーメンターで４２時間培養した。それぞれの培養液約１７０ｌから菌体を回収し、実験２の方法に準じて、超音波処理し、その上清を硫安塩析、透析し、イオン交換カラムクロマトグラフィーと疎水カラムクロマトグラフィーし、得られた部分精製酵素標品の性質を調べた。これらの結果を、前述のスルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９の場合とともに表５にまとめた。
【００６９】
【表５】

【００７０】
また、これらの部分精製酵素を用いて、実験４−３の方法に従って、末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質からのトレハロース調製の実験を行ったところ、スルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９由来の耐熱性トレハロース遊離酵素の場合と同様に、末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質からのトレハロースを遊離することが判明した。
【００７１】
以下、本発明の耐熱性トレハロース遊離酵素の製造方法とそれを利用したトレハロース及びそれを含む糖質の製造方法を実施例Ａで、トレハロース及びそれを含む糖質を含有せしめた組成物を実施例Ｂで示す。
【００７２】
【実施例Ａ−１】
スルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９を実験１の方法に準じて、ファーメンターで約４２時間培養した。培養後、ＳＦ膜を用いて菌体を濃縮し、約５ｌの菌体懸濁液を回収し、更に、その懸濁液をミニラボで処理し、含まれる菌体を破砕した。処理液を遠心分離し、約４．８ｌの遠心上清を得た。この上清に飽和度約０．７になるように硫安を加え、酵素を塩析し、遠心分離で沈殿物を回収し、１０ｍＭトリス・塩酸緩衝液（ｐＨ８．５）に溶解後、同緩衝液に対して透析した。続いて、同緩衝液で平衡化した三菱化成工業株式会社製ゲル『セパビーズ ＦＰ−ＤＡ１３』を用いたイオン交換カラムクロマトグラフィー（ゲル容量約２ｌ）を５回行った。吸着した酵素を０Ｍから０．５Ｍ食塩濃度のリニアグラジエントで溶出させ、０．１５Ｍ食塩濃度付近で溶出した酵素活性画分を回収した後、ＵＦ膜で濃縮し、耐熱性非還元性糖質生成酵素（３２．６単位／ｍｌ）と耐熱性トレハロース遊離酵素（５８．５単位／ｍｌ）を含む濃縮酵素液約３００ｍｌを回収した。次いで、両酵素活性画分を１Ｍ硫酸アンモニウムを含む同緩衝液に対して透析し、その透析液を遠心分離し、不溶物を除去し、得られる上清をブチルトヨパール ６５０を用いた疎水性カラムクロマトグラフィー（ゲル量３５０ｍｌ）を５回行い、耐熱性非還元性糖質生成酵素と耐熱性トレハロース遊離酵素を分離した。１５％とうもろこし澱粉乳に最終濃度０．１重量％となるように炭酸カルシウムを加えた後、ｐＨ６．０に調整し、これにノボ社製α−アミラーゼ『ターマミール６０Ｌ』を澱粉グラム当たり０．２重量％になるよう加え、９５℃で１５分間反応させた。その反応液をオートクレーブ（２ｋｇ／ｃｍ^２）を３０分間行った後、５８℃に冷却し、ｐＨを５．５に調製し、こ れにイソアミラーゼを澱粉グラム当たり２，０００単位、上記調製の耐熱性非還元性糖質生成酵素を澱粉グラム当たり０．５単位、耐熱性トレハロース遊離酵素を澱粉グラム当たり０．５単位加え、９６時間反応させた。その反応液を９７℃で３０分間保った後、冷却し、瀘過して得られる瀘液を、常法に従って、活性炭で脱色し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度６０％のシラップを固形物当たり約９３％で得た。本品は固形物当たりトレハロースを７１．２％、グルコシルトレハロースを３．０％、マルトシルトレハロースを１．３％、グルコースを２．９％、マルトースを１１．１％、マルトトリオースを８．５％及びマルトテトラオース以上のマルトオリゴ糖を２．０％を含有しており、まろやかで上品な甘味、低い還元性、低い粘度、適度の保湿性を有し、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００７３】
【実施例Ａ−２】
実施例Ａ−１の方法で得られた糖液を原糖液とし、トレハロースの含量を高めるため、東京有機化学工業株式会社製ナトリウム型強酸性カチオン交換樹脂『ＸＴ−１０１６』を用いたカラム分画を行った。樹脂を内径５．４ｃｍのジャケット付ステンレス製カラム４本に充填し、直列につなぎ樹脂層全長２０ｍとした。カラム内温度を５５℃に維持しつつ、糖液を樹脂に対して５ｖ／ｖ％加え、これに５５℃の温水をＳＶ０．１３で流して分画し、グルコース、マルトース及びマルトトリオースなどの夾雑糖類を除去し、トレハロース高含有画分を採取した。更に、精製、濃縮し、真空乾燥し、粉砕して、トレハロース高含有粉末を固形物当たり約５７％で得た。本品はトレハロースを９７％含有しており、極めて低い還元性、まろやかで上品な甘味を有し、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００７４】
【実施例Ａ−３】
実施例Ａ−２方法で得られたトレハロース高含有画分を、常法に従って、活性炭で脱色しイオン交換樹脂により脱塩して精製した溶液を濃度約７０％に濃縮した後、助晶機にとり、種晶としてトレハロース含水結晶約２％を加えて徐冷し、晶出率約４５％のマスキットを得た。本マスキットを乾燥塔上のノズルより１５０ｋｇ／ｃｍ^２の高圧にて噴霧した。これと同時に８５℃の熱風を乾燥塔の上部 より送風し、底部に設けた移送金網コンベア上に結晶粉末を捕集し、コンベアの下より４５℃の温風を送りつつ、該粉末を乾燥塔外に徐々に移動させて、取り出した。この結晶粉末を熟成塔に充填して温風を送りつつ、１０時間熟成させ、結晶化と乾燥を完了し、トレハロース含水結晶粉末を、原料のトレハロース高含有糖液に対して固形物当たり約９０％の収率で得た。本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００７５】
【実施例Ａ−４】
実施例Ａ−２の方法で得られたトレハロース高含有画分を、実施例Ａ−３と同様に精製し、次いで蒸発釜にとり、減圧下で煮詰め、水分約３．０％のシラップとした。次いで助晶機に移し、これに種晶として無水結晶トレハロースをシラップ固形物当たり１％加え、１２０℃で５分間撹拌助晶し、次いで、アルミ製バットに取り出し、１００℃で６時間晶出熟成させてブロックを調製した。次いで、本ブロックを切削機にて粉砕し、流動乾燥して、水分０．３％の無水結晶トレハロース粉末を、原料のトレハロース高含有糖液に対して約８５％の収率で得た。本品は、食品、化粧品、医薬品、その原材料、又は加工中間物などの含水物の脱水剤としてのみならず、上品な甘味を有する白色粉末甘味料としても、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００７６】
【実施例Ａ−５】
スルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９の変異株を実施例Ａ−１の方法に準じて、ファーメンターで約４２時間培養した。培養後、ＳＦ膜を用いて菌体を濃縮し、約５ｌの菌体懸濁液を回収し、更に、その懸濁液をミニラボで処理し、含まれる菌体を破砕した。処理液を遠心分離し、約４．８ｌの遠心上清を得た。この上清に飽和度約０．７になるように硫安を加え、酵素を塩析し、遠心分離で沈殿物を回収し、１０ｍＭリン酸緩衝液（ｐＨ６．５）に溶解後、同緩衝液に対して透析し、耐熱性非還元性糖質生成酵素（約１５単位／ｍｌ）と耐熱性トレハロース遊離酵素（約１２単位／ｍｌ）を含む酵素液約６００ｍｌを回収した。次いで、疎水性カラムクロマトグラフィーを行い耐熱性非還元性糖質生成酵素を５８５０単位、耐熱性トレハロース遊離酵素を３９６０単位回収した。馬鈴薯澱粉１重量部に水６重量部とナガセ生化学工業株式会社製α−アミラーゼ『ネオスピターゼ』０．０１重量部とを加え、撹拌混合し、この懸濁液のｐＨを６．２に調整した後、８５乃至９０℃に保ち、澱粉の糊化・液化を行い、その液化液を１２０℃で１０分間加熱してα−アミラーゼを失活させた後、６０℃に冷却し、ｐＨを５．５に調整し、これに、あらかじめ透析し添加されていたスクロースを除き、ＵＦ膜濃縮したノボ・ノルデイスク・バイオインダストリー株式会社販売プルラナーゼ『プロモザイム ２００Ｌ』を澱粉グラム当たり５００単位、及び上記の方法で調製した耐熱性非還元性糖質生成酵素を澱粉グラム当たり１単位、耐熱性トレハロース遊離酵素を澱粉グラム当たり１単位加え７２時間反応させた。その反応液を９７℃で３０分間して酵素を失活させた後、５０℃、ｐＨ５．０に調整し、ナガセ生化学工業株式会社製グルコアミラーゼ『グルコチーム』を澱粉グラム当たり１０単位加えて２４時間反応させ、次いで加熱して酵素を失活させた。本溶液を、常法に従って、活性炭で脱色し、イオン交換樹脂により脱塩し、濃度約６０％に濃縮した。本糖液中には固形物当たり７９．５％のトレハロースを含有していた。イオン交換樹脂として、オルガノ株式会社販売ナトリウム型強酸性カチオン交換樹脂『Ｃ６０００』を用いた以外は、実施例Ａ−２の方法に従ってカラムクロマトグラフィーを行い、トレハロース高含有画分を採取した。本高含有液は、固形物当たり約９５％のトレハロースを含有していた。本溶液を濃度７５％に濃縮した後、助晶機にとり、種晶としてトレハロース含水結晶約２％を加えて撹拌助晶し、次いで、プラスチック製バットに取り出し、室温で３日間放置し晶出熟成させてブロックを調製した。次いで、本ブロックを切削機にて粉砕してトレハロース含水結晶粉末を、原料澱粉に対して固形物当たり約７０％の収率で得た。本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００７７】
【実施例Ａ−６】
スルフォロブス・ソルファタリカス ＡＴＣＣ３５０９１を実験１の方法に準じて、ファーメンターで約４２時間培養した。培養後、実施例Ａ−１の方法に準じて、ＳＦ膜濃縮し、菌体破砕し、その遠心上清を硫安塩析し、塩析物を透析後、イオン交換カラムクロマトグラフィーを行い、酵素活性画分を回収した後、ＵＦ膜で濃縮し、耐熱性非還元性糖質生成酵素（２６．４単位／ｍｌ）と耐熱性トレハロース遊離酵素（５７．５単位／ｍｌ）を含む濃縮酵素液約１５０ｍｌを回収した。次いで、疎水性カラムクロマトグラフィーを行い、耐熱性非還元性糖質生成酵素２６５０単位と耐熱性トレハロース遊離酵素を５９５０単位回収した。濃度６％の馬鈴薯澱粉乳を加熱糊化させた後、ｐＨ４．５、温度５０℃に調整し、これにイソアミラーゼを澱粉グラム当たり５００単位の割合になるように加え、２０時間反応させた。その反応液をｐＨ６．５に調整し、オートクレーブ（１２０℃）を１０分間行い、次いで９５℃に冷却し、これにノボ社製α−アミラーゼ『ターマミール６０Ｌ』を澱粉グラム当たり０．１％重量部の割合になるよう加え、１５分間反応させた。その反応液をオートクレーブ（１３０℃）を３０分間行った後、６５℃に冷却し、これに上記調製の非還元性糖質生成酵素酵素を澱粉グラム当たり１単位、トレハロース遊離酵素を含む濃縮液を澱粉グラム当たり１単位加え、７２時間反応させた。その反応液を９７℃で３０分間保った後、５０℃、ｐＨ５．０に調整し、グルコチームを澱粉グラム当たり１０単位加えて２４時間反応させ、次いで加熱して酵素を失活させた。本溶液を、常法に従って、活性炭で脱色し、イオン交換樹脂により脱塩し、濃度約６０％に濃縮した。本糖液中には固形物当たり８０．９％のトレハロースを含有していた。本溶液を濃度約８４％に濃縮した後、助晶機にとり、種晶としてトレハロース含水結晶約２％を加えて撹拌助晶し、次いで、プラスチック製バットに取り出し、室温で３日間放置し晶出熟成させてブロックを調製した。次いで、本ブロックを切削機にて粉砕してトレハロース含水結晶粉末を、原料澱粉に対して固形物当たり約９０％の収率で得た。本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００７８】
【実施例Ｂ−１ 甘味料】
実施例Ａ−３の方法で得たトレハロース含水結晶粉末１重量部に、東洋精糖株式会社販売α−グリコシルステビオシド『αＧスイート』０．０１重量部及び味の素株式会社製Ｌ−アスパルチル−Ｌ−フェニルアラニンメチルエステル『アスパルテーム』０．０１重量部を均一に混合し、顆粒成型機にかけて、顆粒状甘味料を得た。本品は、甘味の質が優れ、蔗糖の約２．５倍の甘味度を有し、甘味度当たりカロリーは、蔗糖の約１／２．５に低下している。本甘味料は、それに配合した高甘味度甘味物の分解もなく、安定性に優れており、低カロリー甘味料として、カロリー摂取を制限している肥満者、糖尿病者などのための低カロリー飲食物などに対する甘味付けに好適である。また、本甘味料は、不溶性グルカンの生成も少ないことより、虫歯を抑制する飲食物などに対する甘味付けにも好適である。
【００７９】
【実施例Ｂ−２ ハードキャンディー】
濃度５５％蔗糖溶液１００重量部に実施例Ａ−１の方法で得たトレハロース含有シラップ３０重量部を加熱混合し、次いで減圧下で水分２％未満になるまで加熱濃縮し、これにクエン酸１重量部及び適量のレモン香料と着色料とを混和し、常法に従って成型し、製品を得た。本品は、歯切れ、呈味良好で、蔗糖の晶出も起こらない高品質のハードキャンデーである。
【００８０】
【実施例Ｂ−３ チューインガム】
ガムベース３重量部を柔らかくなる程度に加熱溶融し、これに蔗糖４重量部及び実施例Ａ−３の方法で得たトレハロース含水結晶粉末３重量部とを加え、更に適量の香料と着色料とを混合し、常法に従って、ロールにより練り合わせ、成形、包装して製品を得た。本品は、テクスチャー、風味とも良好なチューインガムである。
【００８１】
【実施例Ｂ−４ 加糖練乳】
原乳１００重量部に実施例Ａ−１の方法で得たトレハロース含有シラップ３重量部及び蔗糖１重量部を溶解し、プレートヒーターで加熱殺菌し、次いで濃度７０％に濃縮し、無菌状態で缶詰して製品を得た。本品は、温和な甘味で、風味もよく、乳幼児食品、フルーツ、コーヒー、ココア、紅茶などの調味用に有利に利用できる。
【００８２】
【実施例Ｂ−５ 乳酸菌飲料】
脱脂粉乳１７５重量部、実施例Ａ−１の方法で得たトレハロース含有シラップ１３０重量部及び特開平４−２８１７９５号公報で開示されているラクトスクロース高含有粉末５０重量部を水１，１５０重量部に溶解し、６５℃で３０分間殺菌し、４０℃に冷却後、これに、常法に従って、乳酸菌のスターターを３０重量部植菌し、３７℃で８時間培養して乳酸菌飲料を得た。本品は、風味良好な乳酸菌飲料である。また、本品は、オリゴ糖を含有し、乳酸菌を安定に保持するだけでなく、ビフィズス菌増殖促進作用をも有する。
【００８３】
【実施例Ｂ−６ 粉末ジュース】
噴霧乾燥により製造したオレンジ果汁粉末３３重量部に対して、実施例Ａ−２の方法で得たトレハロース高含有粉末５０重量部、蔗糖１０重量部、無水クエン酸０．６５重量部、リンゴ酸０．１重量部、Ｌ−アスコルビン酸０．１重量部、クエン酸ソーダ０．１重量部、プルラン０．５重量部、粉末香料適量をよく混合撹拌し、粉砕し微粉末にしてこれを流動層造粒機に仕込み、排風温度４０℃、風量１５０ｍ^３とし、これに、実施例Ａ−１の方法で得たトレハロース含有シラッ プをバインダーとしてスプレーし、３０分間造粒し、計量、包装して製品を得た。本品は、果汁含有率約３０％の粉末ジュースである。また、本品は異味、異臭がなく、長期に安定であった。
【００８４】
【実施例Ｂ−７ カスタードクリーム】
コーンスターチ１００重量部、実施例Ａ−１の方法で得たトレハロース含有シラップ１００重量部、マルトース８０重量部、蔗糖２０重量部及び食塩１重量部を充分に混合し、鶏卵２８０重量部を加えて撹拌し、これに沸騰した牛乳１，０００重量部を徐々に加え、更に、これを火にかけて撹拌を続け、コーンスターチが完全に糊化して全体が半透明になった時に火を止め、これを冷却して適量のバニラ香料を加え、計量、充填、包装して製品を得た。本品は、なめらかな光沢を有し、温和な甘味で美味である。
【００８５】
【実施例Ｂ−８ ういろうの素】
米粉９０重量部に、コーンスターチ２０重量部、蔗糖４０重量部、実施例Ａ−３の方法で得たトレハロース含水結晶粉末８０重量部及びプルラン４重量部を均一に混合してういろの素を製造した。ういろうの素と適量の抹茶と水とを混練し、これを容器に入れて６０分間蒸し上げて抹茶ういろうを製造した。本品は、照り、口当りも良好で、風味も良い。また、澱粉の老化も抑制され、日持ちも良い。
【００８６】
【実施例Ｂ−９ あん】
原料あずき１０重量部に、常法に従って、水を加えて煮沸し、渋切り、あく抜きして、水溶性夾雑物を除去して、あずきつぶあん約２１重量部を得た。この生あんに、蔗糖１４重量部、実施例Ａ−１の方法で得たトレハロース含有シラップ５重量部及び水４重量部を加えて煮沸し、これに少量のサラダオイルを加えてつぶあんをこわさないように練り上げ、製品のあんを約３５重量部得た。本品は、色焼けもなく、舌ざわりもよく、風味良好で、あんパン、まんじゅう、だんご、もなか、氷菓などのあん材料として好適である。
【００８７】
【実施例Ｂ−１０ パン】
小麦粉１００重量部、イースト２重量部、砂糖５重量部、実施例Ａ−２の方法で得たトレハロース含有粉末１重量部及び無機フード０．１重量部を、常法に従って、水でこね、中種を２６℃で２時間発酵させ、その後３０分間熟成し、焼き上げた。本品は、色相、すだちとも良好で適度な弾力、温和な甘味を有する高品質のパンである。
【００８８】
【実施例Ｂ−１１ ハム】
豚もも肉１，０００重量部に食塩１５重量部及び硝酸カリウム３重量部を均一にすり込んで、冷室に１昼夜堆積する。これを水５００重量部、食塩１００重量部、硝酸カリウム３重量部、実施例Ａ−６の方法で得たトレハロース含水結晶粉末４０重量部及び香辛料からなる塩漬液に冷室で７日間漬け込み、次いで、常法に従って、冷水で洗浄し、ひもで巻き締め、燻煙し、クッキングし、冷却包装して製品を得た。本品は、色合いもよく、風味良好な高品質のハムである。
【００８９】
【実施例Ｂ−１２ 粉末ペプチド】
不二製油株式会社製４０％食品用大豆ペプチド溶液『ハイニュートＳ』１重量部に、実施例Ａ−６の方法で得たトレハロース含水結晶粉末２重量部を混合し、プラスチック製バットに入れ、５０℃で減圧乾燥し、粉砕して粉末ペプチドを得た。本品は、風味良好で、プレミックス、冷菓などの製菓用材料としてのみならず、経口流動食、経管流動食などの離乳食、治療用栄養剤などとしても有利に利用できる。
【００９０】
【実施例Ｂ−１３ 粉末味噌】
赤味噌１重量部に実施例Ａ−４の方法で得た無水結晶トレハロース粉末３重量部を混合し、多数の半球状凹部を設けた金属板に流し込み、これを室温下で一夜静置して固化し、雛形して１個当たり約４グラムの固形味噌を得、これを粉砕機にかけて粉末味噌を得た。本品は、即席ラーメン、即席吸物などの調味料として有利に利用できる。また、固形味噌は、固形調味料としてだけでなく味噌菓子などとして利用できる。
【００９１】
【実施例Ｂ−１４ 粉末卵黄】
生卵から調製した卵黄をプレート式加熱殺菌機で６０乃至６４℃で殺菌し、得られる液状卵黄１重量部に対して、実施例Ａ−４の方法で得た無水結晶トレハロース粉末４重量部の割合で混合した後バットに移し、一夜放置して、トレハロース含水結晶に変換させてブロックを調製した。本ブロックを切削機にかけて粉末化し、粉末卵黄を得た。本品は、プレミックス、冷菓、乳化剤などの製菓用材料としてのみならず、経口流動食、経管流動食などの離乳食、治療用栄養剤などとしても有利に利用できる。また、美肌剤、育毛剤などとしても有利に利用できる。
【００９２】
【実施例Ｂ−１５ 化粧用クリーム】
モノステアリン酸ポリオキシエチレングリコール２重量部、自己乳化型モノステアリン酸グリセリン５重量部、実施例Ａ−２の方法で得たトレハロース高含有粉末２重量部、α−グリコシル ルチン１重量部、流動パラフィン１重量部、トリオクタン酸グリセリン１０重量部及び防腐剤の適量を常法に従って加熱溶解し、これにＬ−乳酸２重量部、１，３−ブチレングリコール５重量部及び精製水６６重量部を加え、ホモゲナイザーにかけ乳化し、更に香料の適量を加えて撹拌混合しクリームを製造した。本品は、抗酸化性を有し、安定性が高く、高品質の日焼け止め、美肌剤、色白剤などとして有利に利用できる。
【００９３】
【実施例Ｂ−１６ 粉末薬用人参エキス】
薬用人参エキス０．５重量部に実施例Ａ−４の方法で得た無水結晶トレハロース粉末１．５重量部を混捏した後、バットに移し、２日間放置してトレハロース含水結晶に変換させブロックを調製した。本ブロックを切削機にかけて粉末化し、分級して粉末薬用人参エキスを得た。本品を適量のビタミンＢ１及びビタミンＢ２粉末とともに顆粒成型機にかけ、ビタミン含有顆粒状薬用人参エキスとした。本品は、疲労回復剤、強壮、強精剤などとして有利に利用できる。また、育毛剤などとしても利用できる。
【００９４】
【実施例Ｂ−１７ 固体製剤】
ヒト天然型インターフェロン−α標品（株式会社林原生物化学研究所製）を、常法に従って、固定化抗ヒトインターフェロン−α抗体カラムにかけ、該標品に含まれるヒト天然型インターフェロン−αを吸着させ、安定剤である牛血清アルブミンを素通りさせて除去し、次いで、ｐＨを変化させて、ヒト天然型インターフェロン−αを実施例Ａ−２の方法で得たトレハロース高含有粉末を５％含有する生理食塩水を用いて溶出した。本液を精密濾過し、約２０倍量の株式会社林原商事販売無水結晶マルトース粉末『ファイントース』に加えて脱水、粉末化し、これを打錠機にて打錠し、１錠（約２００ｍｇ）当たりヒト天然型インターフェロン−αを約１５０単位含有する錠剤を得た。本品は、舌下錠などとして、一日当たり、大人１乃至１０錠程度が経口的に投与され、ウイルス性疾患、アレルギー性疾患、リューマチ、糖尿病、悪性腫瘍などの治療に有利に利用できる。とりわけ、近年、患者数の急増しているエイズ、肝炎などの治療剤として有利に利用できる。本品は、トレハロースと共にマルトースが安定剤として作用し、室温でも放置してもその活性を長期間よく維持する。
【００９５】
【実施例Ｂ−１８ 糖衣錠】
重量１５０ｍｇの素錠を芯剤とし、これに実施例Ａ−３の方法で得たトレハロース含水結晶粉末４０重量部、プルラン（平均分子量２０万）２重量部、水３０重量部、タルク２５重量部及び酸化チタン３重量部からなる下掛け液を用いて錠剤重量が約２３０ｍｇになるまで糖衣し、次いで、同じトレハロース含水結晶粉末６５重量部、プルラン１重量部及び水３４重量部からなる上掛け液を用いて、糖衣し、更に、ロウ液で艶出しして光沢の在る外観の優れた糖衣錠を得た。本品は、耐衝撃性にも優れており、高品質を長期間維持する。
【００９６】
【実施例Ｂ−１９ 練歯磨】
配合
第２リン酸カルシウム ４５．０％
プルラン ２．９５％
ラウリル硫酸ナトリウム １．５％
グリセリン ２０．０％
ポリオキシエチレンソルビタンラウレート ０．５％
防腐剤 ０．０５％
実施例Ｂ−３に方法で得たトレハロース含水結晶粉末 １２．０％
マルチトール ５．０％
水 １３．０％
上記の材料を常法に従って混合し、練歯磨を得た。本品は、適度の甘味を有しており、特に子供用練歯磨として好適である。
【００９７】
【実施例Ｂ−２０ 流動食用固体製剤】
実施例Ａ−３の方法で製造したトレハロース含水結晶粉末５００重量部、粉末卵黄２７０重量部、脱脂粉乳２０９重量部、塩化ナトリウム４．４重量部、塩化カリウム１．８重量部、硫酸マグネシウム４重量部、チアミン０．０１重量部、アスコルビン酸ナトリウム０．１重量部、ビタミンＥアセテート０．６重量部及びニコチン酸アミド０．０４重量部からなる配合物を調製し、この配合物２５グラムずつ防湿性ラミネート小袋に充填し、ヒートシールして製品を得た。本品は、１袋分を約１５０乃至３００ｍｌの水に溶解して流動食とし、経口的、又は鼻腔、胃、腸などへ経管的使用方法により利用され、生体へのエネルギー補給用に有利に利用できる。
【００９８】
【実施例Ｂ−２３ 外傷治療用膏薬】
実施例Ａ−３の方法で製造したトレハロース含水結晶粉末２００重量部及びマルトース３００重量部に、ヨウ素３重量部を溶解したメタノール５０重量部を加え混合し、更に１０ｗ／ｖ％プルラン水溶液２００重量部を加えて混合し、適度の延び、付着性を示す外傷治療用膏薬を得た。本品は、ヨウ素による殺菌作用のみならず、トレハロースによる細胞へのエネルギー補給剤としても作用することから、治癒期間が短縮され、創面もきれいに治る。
【００９９】
【発明の効果】
上記から明らかなように、本発明の新規耐熱性トレハロース遊離酵素は、末端にトレハロース構造を有するグルコース重合度が３以上の非還元性糖質からトレハロースを遊離し、熱安定性も優れており、また、還元性澱粉部分分解物に耐熱性非還元性糖質生成酵素とともに作用させることによって、雑菌汚染の少ない５５℃を越える高温で、高収率でトレハロースを生成する。そのトレハロースの分離、精製も容易であり、このようにして得られるトレハロース及びそれを含む糖質は安定性に優れ、良質で上品な甘味を有している。また、経口摂取により消化吸収され、カロリー源となる。トレハロース及びそれを含む糖質は甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【０１００】
従って、本発明の確立は、安価で無限の資源である澱粉に由来する澱粉部分分解物から、従来、望むべくして容易に得られなかったトレハロース及びそれを含む糖質を工業的に大量かつ安価に供給できる全く新しい道を拓くこととなり、それが与える影響の大きさは、澱粉科学、酵素科学、生化学などの学問分野は言うに及ばず、産業界、とりわけ食品、化粧品、医薬品分野は勿論のこと、農水畜産業、化学工業にも及び、これら産業界に与える工業的意義は計り知れないものがある。
【図面の簡単な説明】
【図１】ＤＥＡＥ−トヨパールからの本発明の耐熱性トレハロース遊離酵素と非還元性糖質生成酵素の溶出パターンを示す図である。
【図２】本発明の耐熱性トレハロース遊離酵素の酵素活性に及ぼす温度の影響を示す図である。
【図３】本発明の耐熱性トレハロース遊離酵素の酵素活性に及ぼすｐＨの影響を示す図である。
【図４】本発明の耐熱性トレハロース遊離酵素の安定性に及ぼす温度の影響を示す図である。
【図５】本発明の耐熱性トレハロース遊離酵素の安定性に及ぼすｐＨの影響を示す図である。[0001]
[Industrial applications]
The present invention relates to a thermostable trehalose-releasing enzyme, a method for producing the same, and a use thereof. More specifically, the present invention relates to a trehalose moiety of a nonreducing saccharide having a trehalose structure at a terminal and a degree of polymerization of glucose of 3 or more, and a glycosyl moiety other than the trehalose moiety. A novel thermostable trehalose-releasing enzyme that specifically hydrolyzes the bond between to release trehalose and a method for producing the same, in addition to trehalose produced using the novel thermostable trehalose-releasing enzyme, and The present invention relates to a composition comprising:
[0002]
[Prior art]
Trehalose (α, α-trehalose) has long been known as a non-reducing carbohydrate having glucose as a constituent sugar, and its existence is described in “Advances in Carbohydrate Chemistry”. 18: 201-225 (1963) Academic Press, Inc. (USA) and Applied and Environmental Microbiology, 56, 3213-3215. Applied and Environmental Microbiology, Applied and Environmental Microbiology. As described on page (1990) and the like, microorganisms, mushrooms, insects, and the like are spread over a wide range in a small amount. Because trehalose is a non-reducing saccharide, it does not cause an aminocarbonyl reaction with a substance having an amino group such as an amino acid or a protein, and does not impair the amino acid-containing substance. Therefore, it is expected that trehalose can be used and processed without worrying about browning and deterioration. Therefore, establishment of an industrial production method is desired.
[0003]
As a method for producing trehalose, for example, a method using a microorganism reported in JP-A-50-154485, or a combination of maltose phosphorylase and trehalose phosphorylase proposed in JP-A-58-216695 is used. A method for converting maltose is known. However, in the method using a microorganism, the cells are used as a starting material, and the content of trehalose contained therein is usually 15 w / w% per solid matter (hereinafter, w / w unless otherwise specified in the present specification). % Is abbreviated as%), and the steps of extracting and purifying it are complicated and unsuitable as an industrial production method. In addition, in the methods using maltose phosphorylase and trehalose phosphorylase, both involve glucose phosphate, it is difficult to increase the substrate concentration. The production rate is low, and it is difficult to keep the reaction system of both enzymes stable and to allow the reaction to proceed smoothly, and it has not yet been realized as an industrial production method.
[0004]
In view of such a situation, the present inventors have conducted an extensive search for an enzyme that produces a saccharide having a trehalose structure from starch sugar, and found that microorganisms such as Rhizobium species M-11 and Arthrobacter species Q36 are capable of glucose polymerization. It has been found that a completely novel enzyme which is conventionally unknown, that is, a non-reducing carbohydrate having a trehalose structure at the terminal is produced from a partially decomposed product of a reducing starch having a degree of 3 or more. Before and after this finding, this non-reducing saccharide can be almost quantitatively converted to trehalose and glucose and / or maltooligosaccharide by trehalose-releasing enzymes also produced by Rhizobium species M-11 and Arthrobacter species Q36. It was found to be hydrolyzed. By using these enzymes in combination, a desired amount of trehalose can be relatively easily obtained using starch as a raw material, and it is expected that the problems relating to trehalose will be completely solved.
[0005]
However, the above enzymes of Rhizobium species M-11 and Arthrobacter species Q36 have poor heat resistance, and when trying to produce trehalose or a non-reducing saccharide having a trehalose structure at a terminal, a temperature of about 55 ° C. or less is required. It is necessary to carry out an enzymatic reaction. In this regard, in "Knowledge of Enzyme Applications," First Edition, pp. 80-129 (1986), "Saccharide-related enzymes" in "Saccharide-related enzymes and their applications," If the temperature is 55 ° C. or lower, there is a danger of various bacterial contamination, and the pH drops during the saccharification reaction. ” In this case, there is a concern that the pH of the reaction solution is lowered due to bacterial contamination and the enzyme is deactivated during the reaction. Therefore, it may be necessary to prevent bacterial contamination by adding lysozyme or the like and adjust the pH of the reaction solution. In addition, when the hydrolysis rate of the partially degraded starch is low, there is a concern that an insoluble product may be formed due to aging of the starch.
[0006]
On the other hand, since the enzyme reaction of the heat-resistant enzyme proceeds even at a high reaction temperature, the reaction using the heat-resistant enzyme is less likely to cause microbial contamination, and it is considered that aging of the partially degraded starch does not easily occur. As a source of the thermostable enzyme, thermophilic bacteria can generally be mentioned. Regarding trehalose production by thermophilic bacteria, "Biotechnology Letters", Vol. 12, pp. 431-432 (1990) and "Biotech Forum Europe", Vol. 201-203 (1991), partially purified enzymes of cells and extract of cells of Sulfolobus solfataricus ATCC 49155 are used to convert glucose and amylose from substrate amylose and soluble starch. It is reported to produce trehalose. However, the enzyme has not been purified, the physicochemical properties shown are inadequate, the mechanism of action is unknown, and merely indicates that trehalose is produced. Therefore, it is desired to establish a new method for producing trehalose by using a thermostable enzyme capable of performing an enzyme reaction at a temperature exceeding 55 ° C.
[0007]
[Problems to be solved by the invention]
It is an object of the present invention to treat trehalose from a partially decomposed product of reducing starch using a thermostable trehalose-releasing enzyme capable of performing an enzymatic reaction under a temperature exceeding 55 ° C. An object of the present invention is to provide a novel method for producing a saccharide, trehalose, or a saccharide containing the trehalose, and a use thereof.
[0008]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have realized a completely new thermostable enzyme having a mechanism of action of releasing trehalose from a non-reducing saccharide having a terminal trehalose structure and a glucose polymerization degree of 3 or more. In hopes of a wide variety of microorganisms producing this enzyme, including thermophilic bacteria, have been widely searched.
[0009]
As a result, the specification of Japanese Patent Application No. 6-166011 is disclosed. (JP-A-8-66188) And heat-resistant non-reducing saccharide-forming enzyme-producing microorganisms Sulfolobus acidocaldarius ATCC 33909 and ATCC 49426, as well as Sulfolobus solfatatricus (Sulfolotubus 91 and SulfolATCC 91). It has been found that it also produces a novel thermostable trehalose-releasing enzyme capable of reacting at a temperature exceeding 55 ° C. By reacting the heat-resistant non-reducing carbohydrate-forming enzyme with this novel heat-resistant trehalose-releasing enzyme on the partially decomposed starch, it is possible to easily carry out the trehalose-forming reaction at a high reaction temperature aimed at. Further, by reacting a heat-resistant non-reducing saccharide-forming enzyme and a novel heat-resistant trehalose-releasing enzyme on the partially decomposed reducing starch and then reacting with glucoamylase, a reaction solution containing a higher-purity trehalose can be obtained. Thus, the present inventors have found that trehalose can be easily produced, and the present invention has been completed.
[0010]
In the present invention, not only the above bacteria, but also belongs to the genus Sulfolobus, and has a trehalose structure at its terminal, and has a specific degree of binding between the trehalose moiety of the non-reducing saccharide having a degree of polymerization of 3 or more and the other glycosyl moiety. Other strains producing a thermostable trehalose-releasing enzyme that hydrolyzes to form trehalose, and mutants of these strains, etc. can also be used as appropriate.
[0011]
The medium used for culturing the microorganism of the present invention may be any nutrient medium capable of growing the microorganism and producing the thermostable trehalose-releasing enzyme of the present invention, and may be either a synthetic medium or a natural medium. The carbon source may be any substance that can be assimilated by microorganisms, for example, glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, carbohydrates such as partially decomposed starch, or citric acid, succinic acid, etc. Organic acids or salts thereof can also be used. The concentration of these carbon sources in the medium is appropriately selected depending on the type of the carbon source. For example, in the case of partially degraded starch, the content is usually preferably 20% or less, and more preferably 5% or less in view of the growth and proliferation of bacteria. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract, and meat extract. As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt and the like are appropriately used.
[0012]
The cultivation is usually performed aerobically at a temperature of 40 to 95 ° C, preferably 50 to 90 ° C, at a pH of 1 to 7, preferably 2 to 6. The culturing time may be any time during which the present microorganism can proliferate, and is preferably from 10 hours to 100 hours. The concentration of dissolved oxygen in the culture solution is not particularly limited, but is usually preferably 0.5 to 20 ppm. Therefore, means such as adjusting the amount of ventilation, stirring, adding oxygen to the ventilation, and increasing the pressure in the fermenter are employed. The culture method may be either batch culture or continuous culture.
[0013]
After culturing the microorganism in this way, the enzyme of the present invention is recovered. The present enzyme activity is mainly observed in the cells of the culture, and can be purified and used by a known method. As an example, a crude enzyme preparation obtained by salting out a treated product of a culture solution with ammonium sulfate is dialyzed, and then anion-exchange column chromatography using a gel “DEAE-Toyopearl” manufactured by Tosoh Corporation, followed by an Hydrophobic column chromatography using gel "Butyl Toyopearl" etc., gel filtration chromatography using gel "Toyopearl HW-55" etc., hydrophobic column chromatography using butyl Toyopearl again, Pharmacia Biotech A single enzyme can be obtained electrophoretically by purification using gel filtration chromatography using gel "Super Rose 12" manufactured by Co., Ltd. or the like.
[0014]
The thermostable trehalose-releasing enzyme of the present invention thus obtained has the following physicochemical properties.
(1) Action
Between the trehalose moiety and the other glycosyl moiety of the non-reducing saccharide having a terminal trehalose structure and a glucose polymerization degree of 3 or more
It specifically hydrolyzes the bond.
(2) Molecular weight
Approximately 54,000 to 64,000 by SDS-gel electrophoresis.
Dalton.
(3) Isoelectric point
The pI was about 5.6 to 6.6 by electrophoresis containing ampholine.
(4) Optimal temperature
pH 6.0, 30 minutes reaction, around 75 ° C.
(5) Optimum pH
The reaction is performed at 60 ° C for 30 minutes, and the pH is about 5.5 to 6.0.
(6) Temperature stability
Stable up to around 85 ° C by holding pH 7.0 for 60 minutes.
(7) pH stability
PH of about 4.5 to 9.5 at 25 ° C for 16 hours.
[0015]
The activity of the thermostable trehalose releasing enzyme of the present invention is measured as follows. 1 ml of the enzyme solution is added to 4 ml of 1.25 w / v% maltotriosyltrehalose (also called α-maltotetraosyl α-glucoside) (50 mM phosphate buffer, pH 6.0) as a substrate, and the mixture is reacted at 60 ° C. for 30 minutes. After that, the reaction is stopped by adding a somogi copper solution, and the reducing power is measured by the somogi Nelson method. As a control, the same measurement is performed using an enzyme solution which has been inactivated by heating at 100 ° C. for 30 minutes in advance. Using the measurement method described above, the amount of the enzyme that increases the reducing power corresponding to 1 μmole of glucose per minute is defined as one unit.
[0016]
The substrate of the present enzyme may be any non-reducing saccharide having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more. For example, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose Glycosyl trehalose such as glucosyl trehalose, maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose, which can be obtained by allowing non-reducing saccharide-forming enzyme to act on aose or the like, is used. In addition, starch, amylopectin, amylose, etc., partially hydrolyze starch with amylase or acid, etc., to a partially degraded reductant, a non-reducing saccharide-forming enzyme is reacted with a trehalose structure at the terminal. A low-reducing starch partially decomposed product having a non-reducing sugar having a degree of polymerization of glucose of 3 or more is used.
[0017]
Amylase that partially hydrolyzes starch is described in, for example, "Handbook of Amylases and Related Enzymes", (1988) Pergamon Press (Tokyo). Α-amylase, maltopentaose-forming amylase, maltohexaose-forming amylase, and the like. The use of these amylase in combination with a debranching enzyme such as pullulanase and isoamylase can be advantageously carried out.
[0018]
Japanese Patent Application No. 5-349216 discloses a non-reducing saccharide-forming enzyme which produces a non-reducing saccharide having a trehalose structure at its terminal and a glucose degree of polymerization of 3 or more from a partially decomposed reducing starch. (JP-A-7-143876) Non-reducing carbohydrate-forming enzymes such as Rhizobium species M-11 and Arthrobacter species Q36 can be used. Specification of -166011 (JP-A-8-66188) The heat-resistant non-reducing saccharide-forming enzyme of the genus Sulfolobus disclosed in (1) can be advantageously used.
[0019]
The substrate concentration is not particularly limited. For example, the reaction of the present enzyme proceeds to generate trehalose regardless of whether it is used as a 0.1% substrate solution or a 50% substrate solution. Further, the substrate solution may contain an excessive amount of the substrate that cannot be completely dissolved in the substrate solution. The reaction may be carried out at a temperature at which both enzymes are not inactivated, that is, around 85 ° C, and preferably in the range of 55 to 70 ° C. The reaction pH may be usually adjusted in the range of 4 to 10, but is preferably adjusted in the range of about 5 to 7. The reaction time is appropriately selected according to the progress of the enzyme reaction.
[0020]
A process for producing trehalose according to the present invention using a partially decomposed reductive starch having a degree of polymerization of glucose of 3 or more as a substrate is disclosed in Japanese Patent Application No. 5-349216. (JP-A-7-143876) , Ie, the amount of trehalose production is significantly increased as compared with the reaction solution obtained by the reaction of non-reducing saccharide-forming enzyme with glucoamylase. That is, while the trehalose production rate obtained by the reaction between the non-reducing saccharide-forming enzyme of the prior application and glucoamylase is about 30%, the non-reducing saccharide-forming enzyme of the present invention and the trehalose-releasing enzyme are different from each other. The co-acting reaction has a trehalose production rate of about 60% or more.
[0021]
The principle of this operation is as follows. That is, first, one molecule of a partially degradable reducing starch having a degree of glucose polymerization of 3 or more is converted into one molecule of a non-reducing saccharide having a trehalose structure at a terminal by a non-reducing saccharide-forming enzyme. The reducing saccharide produces one molecule of trehalose and one molecule of partially decomposed reducing starch in which the degree of polymerization of glucose is reduced by 2 by the hydrolysis reaction of trehalose releasing enzyme. If the degree of glucose polymerization of the newly generated partially reduced reductive starch is 3 or more, the partially reduced reductive starch is further converted to a non-reducing sugar having a trehalose structure at a terminal by a non-reducing saccharide-forming enzyme. Trehalose-releasing enzyme to form one molecule of trehalose and a partially decomposed reductive starch. By repeating the reaction of the non-reducing saccharide-forming enzyme and the reaction of trehalose-releasing enzyme, a plurality of molecules of trehalose can be produced from one molecule of the partially degraded starch.
[0022]
The method of this action is to simultaneously react the non-reducing saccharide-forming enzyme and the trehalose-releasing enzyme of the present invention on the partially decomposed reductive starch having a degree of polymerization of glucose of 3 or more. First, a non-reducing saccharide-forming enzyme is allowed to act on the substance, and then trehalose-releasing enzyme is allowed to act. If necessary, glucoamylase may be further acted on to increase the trehalose content.
[0023]
The reaction solution is filtered, centrifuged, and the like to remove insoluble matter, then decolorized with activated carbon, desalted with an H-type or OH-type ion exchange resin, and concentrated to obtain a syrup-like product. Furthermore, it is optional to dry it into a powdery product. If necessary, further purification is optional. For example, fractionation by ion exchange column chromatography, fractionation by activated carbon column chromatography, fractionation by silica gel column chromatography, fractionation with organic solvents such as alcohol and acetone, and decomposition and removal of reducing saccharides by alkali treatment By purifying, it is also easy to obtain a high-purity trehalose product.
[0024]
The saccharide containing trehalose of the present invention thus obtained is hydrolyzed with α-amylase, β-amylase, glucoamylase, α-glucosidase, trehalase, etc., or cyclomaltodextrin / glucanotransferase, if necessary. Glucosyltransferase, etc. to adjust the sweetness, reducing power, etc., and reduce the viscosity. Further processing such as disappearing is optional. Glucose is removed by the above-mentioned purification method, for example, ion exchange column chromatography, and a trehalose-rich fraction is collected. This can be advantageously carried out by purifying and concentrating the product to obtain a syrup-like product, or by further concentrating it to supersaturation and crystallization to obtain trehalose-containing crystals or anhydrous trehalose crystals.
[0025]
As the ion-exchange column chromatography, a salt-type strongly acidic cation exchange resin disclosed in JP-A-58-23799, JP-A-58-72598 and the like is used. Ruka A method of removing contaminating saccharides and collecting a trehalose-rich fraction by ram chromatography can be advantageously carried out. At this time, it is optional to adopt any of the fixed bed system, the moving bed system, and the simulated moving bed system.
[0026]
In order to produce trehalose hydrated crystals, for example, a trehalose-containing solution having a purity of 60% or more and a concentration of 65 to 90% is placed in an auxiliary crystal can, and, if necessary, in the presence of 0.1 to 20% of a seed crystal at a temperature of 95 ° C. Hereinafter, desirably, the mixture is gradually cooled with stirring at a temperature in the range of 10 to 90 ° C. to produce a mass kit containing trehalose hydrated crystals. It is also advantageous to employ a continuous crystallization method in which crystallization is performed while concentrating under reduced pressure. As a method for producing trehalose hydrated crystals or honey-containing crystals containing the same from a mass kit, for example, a known method such as a honey separating method, a block crushing method, a fluid granulation method, and a spray drying method may be employed.
[0027]
In the case of the honey separation method, the mass kit is usually placed in a basket-type centrifugal separator to separate the trehalose hydrated crystals from the nectar (mother liquor), and it is also easy to wash the crystals by spraying a small amount of cold water, if necessary. This method is suitable for producing higher purity trehalose hydrated crystals. In the case of the spray drying method, usually, a mass kit having a concentration of about 60 to 85% and a crystallization rate of about 20 to 60% is sprayed from a nozzle with a high-pressure pump, and hot air at a temperature at which the crystal powder is not dissolved, for example, 60 to 100 ° C. And then aged for about 1 to 20 hours with warm air at 30 to 60 ° C., non-hygroscopic or hardly hygroscopic honey-containing crystals can be easily produced. In the case of the block pulverization method, usually, a mass kit having a water content of about 10 to 20% and a crystallization rate of about 10 to 60% is allowed to stand for several hours to 3 days to crystallize and solidify the whole in the form of a block. Alternatively, if powdered and dried by a method such as cutting, a non-hygroscopic or hardly hygroscopic honey-containing crystal can be easily produced. In order to produce anhydrous crystalline trehalose, hydrated trehalose crystals can be converted by drying. However, in general, a high-concentration trehalose-rich solution having a water content of less than 10% is placed in an auxiliary crystal can and mixed with seed crystals. A mass kit containing anhydrous crystalline trehalose is produced while stirring at a temperature of 50 to 160 ° C., preferably 80 to 140 ° C. under a high temperature drying condition, for example, a block pulverization method, a fluid granulation method. It is produced by crystallization and powdering by a method such as spray drying.
[0028]
The trehalose of the present invention thus produced has no reducing power, is stable, and can be mixed and processed with other materials, particularly amino acids, oligopeptides, proteins and other substances containing amino acids or amino groups. It does not cause browning, generate an unpleasant odor, or damage other mixed materials. In addition, it itself has a good and elegant sweetness. Furthermore, trehalose is easily decomposed into glucose by trehalase, so that it is digested and absorbed by oral ingestion and used as a calorie source. It can be used as a sweetener that is hardly fermented by caries-inducing bacteria or the like and hardly causes caries.
[0029]
In addition, the trehalose of the present invention is used parenterally as a tube feeding agent, an infusion solution, and the like, and is well metabolized and used without concern about toxicity and side effects, and can be advantageously used for replenishing energy to a living body. it can. In addition, in the case of a trehalose hydrated crystal product, because it is a stable sweetener, it can be advantageously used as a sugar coating for tablets in combination with a binder such as pullulan, hydroxyethyl starch, or polyvinylpyrrolidone. In addition, it has properties such as osmotic pressure controllability, shaping properties, shine imparting properties, moisturizing properties, viscosity, anti-crystallization properties of other sugars, poor fermentability, and anti-starch properties.
[0030]
Therefore, the trehalose of the present invention and a saccharide containing the same can be used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, etc. in various compositions such as foods and drinks, foods, feeds, cosmetics, and pharmaceuticals. It can be used advantageously.
[0031]
The trehalose of the present invention and a saccharide containing the trehalose can be directly used as a seasoning for sweetening. If necessary, for example, powdered candy, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L It may be used in admixture with one or more suitable amounts of other sweeteners such as phenylalanine methyl ester, saccharin, glycine, alanine and the like, and if necessary, such as dextrin, starch, lactose etc. It can also be used in combination with other bulking agents.
[0032]
The trehalose and the saccharide powder or crystalline product containing the same according to the present invention may be used as they are, or, if necessary, mixed with a bulking agent, an excipient, a binder, etc., to give granules, spheres, short rods. It can be optionally used by molding into various shapes such as plate, cube, tablet and the like. In addition, the sweetness of the trehalose of the present invention and saccharides containing the same is in good harmony with various substances having other tastes such as sourness, salty taste, astringency, umami, and bitterness, and has high acid resistance and heat resistance. Therefore, it can be advantageously used for sweetening and taste improvement of general foods and drinks, quality improvement, and the like.
[0033]
For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle, mayonnaise, dressing, vinegar, three tablespoon vinegar, powdered sushi vinegar, Chinese ingredients, tentsuyu, noodle soup, sauce, ketchup, grilled meat sauce, curry roux, stew It can be advantageously used as a variety of cooking ingredients such as no-no-miso, soup-no-maki, dashi-no-miso, complex seasonings, mirin, new mirin, table sugar, coffee sugar and the like.
[0034]
In addition, for example, various types of Japanese confectionery such as rice crackers, hail, rice bran, rice cakes, steamed buns, sea cucumber, sea cucumber, sheep shrimp, mizu-yori, nishikidama, jelly, castella, candy, bread, biscuit, crackers, cookies, pies , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy, etc., ice cream, sherbet, etc., ice confectionery, fruit syrup pickling, ice honey, etc., flower Pastes such as paste, peanut paste, fruit paste, spread, jams, marmalade, syrup pickles, fruits such as citrus fruits, processed foods of vegetables, pickles such as Fukujin pickles, betta pickles, senmai pickles, lucky pickles, takuan pickles, Pickles such as Chinese cabbage pickles Livestock meat products such as raw materials, ham, sausage, etc., fish meat ham, fish meat sausage, fish meat products such as kamaboko, chikuwa, tempura, various delicacies such as sea urchin, salted squid, vinegar konbu, sasakisame, fugumi phosphorus dried, seaweed Foods such as tsukudani, wild vegetables, sardines, small fish, shellfish, boiled beans, potato salad, konbu-maki, etc., dairy products such as yogurt, cheese, fish meat, animal meat, fruits, vegetable bottles, canned foods Alcoholic beverages such as liquor, sake, synthetic liquor, liqueurs, and Western liquors; soft drinks such as coffee, cocoa, juice, carbonated beverages, lactic acid beverages, and lactic acid beverages; pudding mixes; hot cake mixes; instant foods such as instant soups and instant soups; Further, it can be advantageously used for sweetening various foods such as baby foods, therapeutic foods and drinks, for improving taste, and for improving quality.
[0035]
In addition, it can be used for raising the preference of feeds, feeds, and the like for livestock, poultry, and other bred animals such as bees, silkworms, and fish. In addition, various solids such as tobacco, toothpaste, lipstick, lip balm, oral liquid, tablets, troches, liver oil drops, mouth fresheners, mouth flavors, gargles, etc. Can be advantageously used as a sweetener for various compositions, as a taste improver, a flavor enhancer, and as a quality improver.
[0036]
As a quality improving agent and a stabilizer, it can be advantageously applied to various physiologically active substances which easily lose their active ingredients and activities, or health foods and pharmaceuticals containing the same. For example, lymphokine-containing liquids such as interferon-α, interferon-β, interferon-γ, Tsumore necrosis factor-α, Tsumore necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, interleukin II, etc. , Hormone-containing solution such as insulin, growth hormone, prolactin, erythropoietin, oocyte stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, pox seedling, tetanus toxoid, hub antitoxin, biological preparations such as human immunoglobulin Solution, antibiotic-containing solution such as penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate, thiamine, riboflavin, L-asco Vitamin-containing liquids such as rubic acid, liver oil, carotenoids, ergosterol, tocopherol, etc., enzyme-containing liquids such as lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucanase, lactase, ginseng extract, terrapin extract, chlorella Extracts, aloe extract, propolis extract and other extracts, viruses, lactic acid bacteria, live bacteria such as yeast, various biologically active substances such as royal jelly, without losing their active ingredients, activity, and stable and high quality health foods Drugs and the like can be easily manufactured.
[0037]
The method for incorporating trehalose into the various compositions as described above may be included in the process until the product is completed, for example, mixing, dissolving, melting, dipping, penetrating, spraying, applying, coating, spraying. Known methods such as injection, crystallization, and solidification are appropriately selected. The amount is usually 0.1% or more, preferably 1% or more.
[0038]
Next, the present invention will be described more specifically by experiments.
[0039]
[Experiment 1 Enzyme production]
Peptone 0.1 w / v%, yeast extract 0.1 w / v%, ammonium sulfate 0.2 w / v%, monopotassium phosphate 0.05 w / v%, magnesium sulfate heptahydrate 0.02 w / v, potassium chloride 0 About 100 ml of a liquid medium containing 0.02 w / v% and water was put into a 500 ml Erlenmeyer flask, sterilized in an autoclave at 120 ° C. for 20 minutes, cooled, and adjusted to pH 3.0 with sulfuric acid. This liquid medium was inoculated with Sulfolobus acidocardarius ATCC 33909, and cultured at 75 ° C. and 130 rpm for 24 hours to obtain a primary seed culture. About 5 l of a medium having the same composition as that of the primary seed culture is put into a fermenter having a volume of 10 l, sterilized, cooled to pH 3.0 and a temperature of 75 ° C., and then 1 v / v% of the primary seed culture is added. The inoculum was incubated at a temperature of 75 ° C. and aerated at 500 ml / min for about 48 hours to obtain a secondary seed culture. About 250 liters of a medium having the same composition as in the case of the primary seed culture is put into a fermenter having a capacity of 300 liters, sterilized, cooled to pH 3.0 and a temperature of 75 ° C., and then 1 v / v% of the secondary seed culture is added. After inoculation, aeration culture was performed at a temperature of 75 ° C. and an aeration rate of 100 l / min for about 42 hours. The enzyme activity of the thermostable trehalose releasing enzyme in the culture solution was about 0.03 unit / ml.
[0040]
[Experiment 2 Purification of enzyme]
About 170 l of the culture solution obtained by the method of Experiment 1 was centrifuged, and 258 g of the contained cells were collected as a wet weight. After 300 ml of 10 mM phosphate buffer (pH 7.0) was added to the cells and suspended, the cells were crushed with an ultrasonic crusher model “US300” manufactured by Nippon Seiki Seisakusho. The crushed liquid was centrifuged (10,000 rpm, 30 minutes) to obtain about 300 ml of a centrifuged supernatant. Ammonium sulfate was added to and dissolved in the solution to a saturation degree of 0.7, left at 4 ° C. for 24 hours, and then centrifuged to collect a salted-out product. The obtained salted-out product was dissolved in a 10 mM Tris / hydrochloric acid buffer (pH 8.5), and dialyzed against the same buffer for 24 hours, and centrifuged to remove insolubles. The dialysate (about 600 ml) was divided into two portions and subjected to ion exchange column chromatography using DEAE-Toyopearl (gel amount: about 350 ml).
[0041]
Both the thermostable trehalose-releasing enzyme and the thermostable non-reducing saccharide-forming enzyme of the present invention are adsorbed to DEAE-Toyopearl, and both enzymes are eluted from the column with the same buffer containing sodium chloride at a concentration of about 0.1 M sodium chloride. Collected as the active fraction.
[0042]
Both enzymatically active fractions were dialyzed against the same buffer containing 1 M ammonium sulfate, and the dialysate was centrifuged to remove insolubles. The resulting supernatant was subjected to gel "butyl toyopearl 650" manufactured by Tosoh Corporation. Hydrophobic column chromatography (gel amount: 350 ml) was performed. When the adsorbed present enzyme was eluted from the column with a linear gradient of 1M to 0M ammonium sulfate concentration, the enzyme was eluted at a different ammonium sulfate concentration from the thermostable trehalose releasing enzyme and the thermostable non-reducing saccharide-forming enzyme. The elution pattern from butyl toyopearl is shown in FIG. The thermostable non-reducing saccharide-forming enzyme elutes at an ammonium sulfate concentration of about 0.8M, and the thermostable trehalose-releasing enzyme elutes at an ammonium sulfate concentration of about 0.2M, and collects the respective enzyme active fractions. Was purified.
[0043]
The thermostable non-reducing saccharide-forming enzyme fraction was dialyzed against the same buffer containing 0.2 M sodium chloride, the dialysate was centrifuged to remove insolubles, and the resulting supernatant was subjected to Sepracol gel "Ultrogel". Gel filtration chromatography using AcA 44 "(gel amount: 350 ml) was performed to collect an enzyme active fraction. Subsequently, the buffer was dialyzed and then subjected to ion exchange column chromatography (gel amount: 10 ml) using a gel "Mono Q" manufactured by Pharmacia LKB to remove the adsorbed enzyme from 0 M to 0.2 M salt. The fraction was eluted from the column with a linear gradient of concentration, and a heat-resistant non-reducing saccharide-forming enzyme active fraction eluted at around 0.1 M salt concentration was collected.
[0044]
For purification of the thermostable trehalose-releasing enzyme, gel filtration chromatography using Toyopearl HW-55 was performed using the thermostable trehalose-releasing enzyme active fraction eluted from butyl toyopearl, and the enzyme-active fraction was recovered. Subsequently, hydrophobic column chromatography using butyl toyopearl 650 was performed again. Further, gel filtration chromatography was performed using a Pharmacia column “Super Rose 12HR 10/30” to collect a heat-resistant trehalose-releasing enzyme active fraction.
[0045]
Throughout the present invention, the activity of the non-reducing saccharide-forming enzyme is represented by an activity value (unit) measured by the following method, unless otherwise specified. That is, 4 ml of 50 mM acetate buffer (pH 5.5) containing 1.25% (w / v) maltopentaose was added, 1 ml of the enzyme solution was added thereto, and the mixture was incubated at 60 ° C. for 60 minutes to react. The reaction is stopped by heating the reaction at 100 ° C. for 100 minutes. After diluting the reaction solution 10 times with distilled water, the reducing power is measured by the Somogi-Nelson method. One unit of the non-reducing saccharide-forming enzyme is defined as the amount of the enzyme that reduces the reducing power corresponding to 1 μmol of maltopentaose per minute under the above conditions.
[0046]
The enzyme activity, specific activity and yield in each step of the purification are shown in Table 1 for the thermostable non-reducing saccharide-forming enzyme and Table 2 for the thermostable trehalose releasing enzyme of the present invention.
[0047]
[Table 1]

[0048]
[Table 2]

[0049]
The purified heat-resistant non-reducing saccharide-forming enzyme and the purified heat-resistant trehalose-releasing enzyme obtained as gel filtration eluates in the steps of Tables 1 and 2, respectively, are used as polyacrylamide gels (gel concentration 7.5%). When the purity was assayed by electrophoresis, the protein band was single and was a highly pure sample.
[0050]
[Experiment 3 physicochemical properties]
[Experiment 3-1 Properties of thermostable trehalose releasing enzyme]
The purified heat-resistant trehalose-releasing enzyme obtained by the method of Experiment 2 was subjected to an electrophoresis method using SDS-polyacrylamide gel (gel concentration: 10%), and a molecular weight marker simultaneously migrated (manufactured by Nippon Bio-Rad Laboratories, Inc.) The molecular weight of the enzyme was measured to be about 54,000 to 64,000 daltons.
[0051]
The purified enzyme was subjected to isoelectric focusing using a polyacrylamide gel (containing 2% ampholine, manufactured by Pharmacia LKB). After the electrophoresis, the pH of the gel was measured to determine the isoelectric point of the enzyme. However, the isoelectric point was about 5.6 to 6.6.
[0052]
The effects of temperature and pH on the activity of this enzyme were examined in accordance with the activity measurement method. The results are shown in FIG. 2 (effect of temperature) and FIG. 3 (effect of pH). The optimum temperature of the enzyme was about 75 ° C. for a reaction of pH 6.0 for 30 minutes, and the optimum pH was about 5.5 to 6.0 for a reaction of 60 ° C. for 30 minutes. The temperature stability of the present enzyme was determined by maintaining the enzyme solution (containing 50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water, and measuring the remaining enzyme activity. The pH stability was determined by keeping the enzyme in a 50 mM buffer at each pH at 25 ° C. for 16 hours, adjusting the pH to 7, and measuring the remaining enzyme activity. The respective results are shown in FIG. 4 (temperature stability) and FIG. 5 (pH stability). The temperature stability of this enzyme was up to about 85 ° C, and the pH stability was 5.5 to 9.5.
[0053]
When the N-terminal amino acid sequence of the purified enzyme was analyzed from the N-terminal to 10 residues using a protein sequencer model “473A” sold by Applied Biosystems Japan, the N-terminal amino acid sequence of the enzyme was methionine-phenylalanine. -Serine-phenylalanine-glycine-glycine-asparagine-isoleucine-glutamic acid-lysine.
[0054]
[Experiment 3-2 Physicochemical properties of thermostable non-reducing saccharide-forming enzyme]
The purified thermostable non-reducing saccharide-forming enzyme obtained by the method of Experiment 2 was subjected to an electrophoresis method using an SDS-polyacrylamide gel (gel concentration: 10%), and compared with the molecular weight marker migrated at the same time, the enzyme of the present enzyme was used. The molecular weight was measured to be about 69000 to 79,000 daltons. The purified enzyme was subjected to isoelectric focusing using a polyacrylamide gel. After the electrophoresis, the pH of the gel was measured to determine the isoelectric point of the enzyme. The isoelectric point was about 5.4 to 6. It was 4.
[0055]
The effects of temperature and pH on the activity of this enzyme were examined in accordance with the activity measurement method. The optimum temperature of the enzyme was about 75 ° C., and the optimum pH was about 5.0 to 5.5. When the temperature stability and the pH stability of the present enzyme were examined in the same manner as in the method of Experiment 3-1, the temperature stability of the present enzyme was up to about 85 ° C, and the pH stability was 4.0 to 9.5. there were.
[0056]
The N-terminal amino acid sequence of the purified enzyme was examined in the same manner as in Experiment 3-1. The N-terminal amino acid sequence of the purified enzyme was methionine-isoleucine-serine-alanine-threonine-tyrosine-arginine-leucine-glutamine-leucine. Met.
[0057]
[Experiment 4 Production of trehalose by thermostable trehalose-releasing enzyme]
[Experiment 4-1 Preparation of non-reducing saccharide-forming enzyme]
Maltose 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v), disodium hydrogen phosphate 0.1% and phosphorus in a 500 ml Erlenmeyer flask 100 ml of a liquid medium (pH 7.0) containing 0.1% of potassium dihydrogenate was taken, and sterilized by autoclaving at 120 ° C. for 20 minutes. After cooling, Rhizobium species M-11 (FERM BP-4130) was inoculated into the liquid medium in the Erlenmeyer flask and seed-cultured at 27 ° C. for 24 hours under rotary shaking. Separately, 20 l of a liquid medium of the same composition is placed in a 30-liter fermenter, sterilized, inoculated with 1 v / v% of the seed culture solution obtained above, and aerated at 30 ° C. for 72 hours while maintaining the liquid medium at pH 6 to 8. Agitation culture was performed. The culture was treated with an ultra-high pressure cell crusher “Minilab” manufactured by Dainippon Pharmaceutical Co., Ltd. to crush the contained cells. After removing insoluble matters by centrifugation, ammonium sulfate fractionation, ion exchange column chromatography using DEAE-Toyopearl, hydrophobic column chromatography using butyltoyopearl, and gel filtration column chromatography using Toyopearl HW-55 were performed. As a result, a non-reducing saccharide-forming enzyme having a specific activity of 195 units / mg protein was obtained in a yield of about 220 units per liter of culture.
[0058]
The activity of the non-reducing saccharide-forming enzyme derived from Rhizobium sp. M-11 was measured by changing 50 mM acetate buffer (pH 5.5) to 50 mM phosphate buffer (pH 7.0) among the measurement conditions of Experiment 2. The reaction temperature was changed from 60 ° C to 40 ° C.
[0059]
[Experiment 4-2 Preparation of non-reducing saccharide having a trehalose structure at the terminal and a glucose polymerization degree of 3 or more]
Non-reducing saccharide-forming enzyme obtained by the method of Experiment 4-1 in a 20% aqueous solution of a partially decomposed reducing starch selected from maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose Was added at a ratio of 2 units per gram of the substrate solid substance, and the mixture was allowed to act at 40 ° C. and pH 7.0 for 48 hours, then deactivated by heating, filtered, decolorized, desalted and concentrated according to a conventional method. Ion exchange column chromatography using a sodium-type strongly acidic cation exchange resin “XT-1016” (degree of crosslinking: 4%) manufactured by Kogyo Co., Ltd. was performed. The resin is packed in three stainless steel columns with a jacket of 2.0 cm in inner diameter and 1 m in length, connected in series, and the reaction sugar solution is added at 5 v / v% to the resin while maintaining the temperature in the column at 55 ° C. Then, hot water at 55 ° C. was flowed at SV 0.13 to fractionate the fraction, and a fraction having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more and a purity of 95% or more of a non-reducing saccharide was recovered. Sodium hydroxide was added to the collected fraction to a concentration of 0.1 N, and the mixture was heated at 100 ° C. for 2 hours to decompose the remaining reducing saccharide. The solution was decolorized with activated carbon, desalted with H-form, OH-form, ion-exchange resin, and α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltosyltrehalose having a purity of 99.0% or more. Non-reducing saccharide preparations of maltotetraosyltrehalose and α-maltopentaosyltrehalose were prepared.
[0060]
[Experiment 4-3 Production of trehalose from non-reducing saccharide by thermostable trehalose-releasing enzyme]
A 5% aqueous solution of the five non-reducing saccharides obtained by the method of Experiment 4-2 was prepared, and the thermostable trehalose-releasing enzyme obtained in Experiment 2 was respectively added at a ratio of 2 units per gram of the solid substrate. In addition, after reacting at 60 ° C. and pH 5.5 for 48 hours, desalting was performed, and the reaction product was analyzed by high performance liquid chromatography using a column “Wakobeads WB-T-330” manufactured by Wako Pure Chemical Industries, Ltd. did. As a control, heat-resistant trehalose-releasing enzyme was allowed to act on maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose in the same manner, and analyzed by high-performance liquid chromatography. Table 3 shows the results.
[0061]
[Table 3]

[0062]
As is clear from the results in Table 3,
(1) The thermostable trehalose-releasing enzyme specifically hydrolyzes the bond between the trehalose moiety and the glycosyl moiety of a non-reducing saccharide having a trehalose structure at the terminal and having a degree of polymerization of 3 or more, and thus trehalose and glucose And a reducing saccharide having a degree of polymerization of 1 or more. (2) Maltooligosaccharides are not affected at all by thermostable trehalose-releasing enzymes.
[0063]
From these results, the thermostable trehalose-releasing enzyme of the present invention has very specific binding between the trehalose moiety of the non-reducing saccharide having a trehalose structure at the terminal and a degree of polymerization of glucose of 3 or more and other glycosyl moieties. It is considered to be an enzyme with a completely new mechanism of action that hydrolyzes to release trehalose.
[0064]
[Experiment 5 Preparation of trehalose from partially decomposed reducing starch]
After gelatinizing the 5% waxy corn starch suspension by heating, the pH is adjusted to 4.5 and the temperature is adjusted to 50 ° C., and isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) is added thereto at a ratio of 4000 units per gram of starch. And reacted for 20 hours. The reaction solution was autoclaved (120 ° C., 10 minutes), cooled to 60 ° C., and subjected to glucose filtration chromatography (gel amount 750 ml) using a column “Toyopearl column HW50” manufactured by Tosoh Corporation. A partially degraded reducing starch having a degree of 37 to 11 was separated.
[0065]
The resulting partially decomposed reducing starch or maltotriose having a glucose polymerization degree of 3 was adjusted to a concentration of 1% with a 10 mM phosphate buffer (pH 5.5), and the purified heat-resistant solution prepared by the method of Experiment 2 was added thereto. A non-reducing saccharide-forming enzyme preparation and a purified heat-resistant trehalose-releasing enzyme preparation were added at a ratio of 4 units per solid substrate, allowed to act at 60 ° C. for 24 hours, and a portion was taken and desalted. The reaction products were analyzed by high performance liquid chromatography. After the remaining reaction solution was further adjusted to 50 ° C. and pH 4.5, glucoamylase (manufactured by Seikagaku Corporation) was added at a ratio of 50 units per substrate solid, allowed to act for 10 hours, and desalted similarly. Then, the reaction product was analyzed by high performance liquid chromatography. Table 4 shows the results.
[0066]
[Table 4]

[0067]
As shown in Table 4, the trehalose production rate after the action of the thermostable non-reducing saccharide-forming enzyme and the thermostable trehalose-releasing enzyme was as low as 2.2% for maltotriose having a glucose polymerization degree of 3. However, a high value of 63.3 to 81.2% was obtained with a partially degraded starch having a degree of polymerization of glucose of 10.8 to 36.8. It was also found that the higher the glucose polymerization degree, the higher the purity of trehalose obtained. Furthermore, it was also found that the purity of the trehalose produced was further increased by decomposing the non-reducing saccharide having a trehalose structure at the terminal remaining with glucoamylase and having a degree of polymerization of 3 or more into trehalose and glucose.
[0068]
[Experiment 6 Production and properties of thermostable trehalose releasing enzyme derived from other Sulfolobus microorganisms]
The fermenter was cultured for 42 hours in the same manner as in Experiment 1, except that Sulfolobus acidocardarius ATCC 33909 was replaced by Sulfolobus acidocardarius ATCC 49426, Sulfolobus solfataricus ATCC 35091, and Sulfolobus solfataricus ATCC 35092. The cells were recovered from about 170 l of each culture, sonicated according to the method of Experiment 2, the supernatant was dialyzed with ammonium sulfate, dialyzed, and subjected to ion exchange column chromatography and hydrophobic column chromatography. The properties of the partially purified enzyme preparation thus obtained were examined. These results are summarized in Table 5 together with the case of Sulfolobus acidocardarius ATCC33909 described above.
[0069]
[Table 5]

[0070]
Using these partially purified enzymes, according to the method of Experiment 4-3, an experiment of preparing trehalose from a non-reducing saccharide having a trehalose structure at the terminal and having a degree of polymerization of glucose of 3 or more was performed. As in the case of the thermostable trehalose releasing enzyme derived from Acidocardarius ATCC33909, it was found that trehalose was released from non-reducing saccharides having a trehalose structure at the terminal and having a degree of polymerization of 3 or more.
[0071]
Hereinafter, a method for producing a thermostable trehalose-releasing enzyme of the present invention and a method for producing trehalose and a saccharide containing the same using the same are described in Example A, and a composition containing trehalose and a saccharide containing the same is described in Example. Shown by B.
[0072]
Example A-1
Sulfolobus acidocardarius ATCC33909 was cultured in a fermenter for about 42 hours according to the method of Experiment 1. After the culture, the cells were concentrated using an SF membrane, about 5 l of the cell suspension was collected, and the suspension was treated in a minilab to disrupt the cells contained. The treated liquid was centrifuged to obtain about 4.8 l of a centrifuged supernatant. Ammonium sulfate was added to the supernatant to a saturation level of about 0.7, the enzyme was salted out, the precipitate was collected by centrifugation, and dissolved in a 10 mM Tris / hydrochloric acid buffer (pH 8.5). The solution was dialyzed. Subsequently, ion-exchange column chromatography (gel volume: about 2 l) was performed 5 times using a gel “Sepabeads FP-DA13” manufactured by Mitsubishi Kasei Kogyo Co., Ltd. equilibrated with the same buffer. The adsorbed enzyme was eluted with a linear gradient of 0M to 0.5M salt concentration, and the enzyme active fraction eluted at around 0.15M salt concentration was collected and concentrated with a UF membrane to produce a heat-resistant non-reducing saccharide. About 300 ml of a concentrated enzyme solution containing the enzyme (32.6 units / ml) and the thermostable trehalose releasing enzyme (58.5 units / ml) was recovered. Next, both enzyme active fractions were dialyzed against the same buffer containing 1 M ammonium sulfate, the dialysate was centrifuged to remove insolubles, and the resulting supernatant was subjected to a hydrophobic column using butyltoyopearl 650. Chromatography (gel amount: 350 ml) was performed five times to separate the thermostable non-reducing saccharide-forming enzyme from the thermostable trehalose releasing enzyme. After adding calcium carbonate to 15% corn starch milk to a final concentration of 0.1% by weight, the pH was adjusted to 6.0, and α-amylase “Termamale 60L” manufactured by Novo was added in an amount of 0.2% per gram of starch. %, And reacted at 95 ° C. for 15 minutes. The reaction solution was placed in an autoclave (2 kg / cm ² ) For 30 minutes and then cooled to 58 ° C to adjust the pH to 5.5, to which 2,000 units of isoamylase were added per gram of starch, and the heat-resistant non-reducing saccharide-forming enzyme prepared above was added. 0.5 units per gram of starch and 0.5 units per gram of the starch were added and reacted for 96 hours. The reaction solution was kept at 97 ° C. for 30 minutes, cooled, filtered, and the filtrate obtained was decolorized with activated carbon and desalted with H-type and OH-type ion exchange resins in accordance with a conventional method. And further concentrated to give a syrup with a concentration of 60% at about 93% per solid. This product contains 71.2% of trehalose, 3.0% of glucosyl trehalose, 1.3% of maltosyl trehalose, 2.9% of glucose, 11.1% of maltose and 8.1 of maltotriose per solid. Contains 5% and 2.0% malto-oligosaccharide of maltotetraose or more, has mellow and elegant sweetness, low reducing property, low viscosity, moderate moisturizing property, sweetener, taste improver, As a quality improving agent, a stabilizer, an excipient and the like, it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.
[0073]
Example A-2
The sugar solution obtained by the method of Example A-1 was used as a raw sugar solution, and in order to increase the content of trehalose, a column using a sodium-type strongly acidic cation exchange resin “XT-1016” manufactured by Tokyo Organic Chemical Industry Co., Ltd. Painting. The resin was packed in four jacketed stainless steel columns having an inner diameter of 5.4 cm and connected in series to make the total length of the resin layer 20 m. While maintaining the temperature in the column at 55 ° C, a sugar solution was added at 5 v / v% to the resin, and hot water at 55 ° C was flowed at SV 0.13 to fractionate the fraction, and glucose, maltose, maltotriose, etc. Contaminant saccharides were removed, and a trehalose-rich fraction was collected. Further purification, concentration, vacuum drying and grinding gave a trehalose-rich powder at about 57% per solid. This product contains 97% trehalose, has extremely low reducing properties, has a mellow and elegant sweet taste, and is used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. It can be advantageously used for various compositions such as cosmetics and pharmaceuticals.
[0074]
Example A-3
The trehalose-rich fraction obtained by the method of Example A-2 was decolorized with activated carbon, desalted with an ion-exchange resin, and purified to a concentration of about 70%. Then, about 2% of hydrated trehalose crystals were added as seed crystals, and the mixture was gradually cooled to obtain a mass kit having a crystallization rate of about 45%. 150kg / cm from the nozzle on the drying tower ² At high pressure. At the same time, hot air of 85 ° C. is blown from the top of the drying tower, and the crystal powder is collected on a transfer wire mesh conveyor provided at the bottom. Moved slowly out and removed. The crystal powder is filled in a maturation tower and aged for 10 hours while sending hot air to complete crystallization and drying. The trehalose-containing hydrated crystal powder is added to the raw material trehalose-rich saccharide solution at about 90% per solid. % Yield. This product has practically no hygroscopicity, is easy to handle, and has various compositions such as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. It can be used to advantage for things.
[0075]
Example A-4
The trehalose-rich fraction obtained by the method of Example A-2 was purified in the same manner as in Example A-3, then placed in an evaporator and boiled down under reduced pressure to obtain a syrup having a water content of about 3.0%. Then, it was transferred to an auxiliary crystallizer, and anhydrous trehalose was added as a seed crystal at a concentration of 1% per syrup solid, and stirred at 120 ° C. for 5 minutes, then taken out in an aluminum vat and crystallized and aged at 100 ° C. for 6 hours. Then, a block was prepared. Next, this block was pulverized by a cutting machine and fluid-dried to obtain anhydrous crystalline trehalose powder having a water content of 0.3% in a yield of about 85% with respect to the raw material trehalose-rich saccharide solution. This product can be used not only as a dehydrating agent for hydrates such as foods, cosmetics, pharmaceuticals, raw materials, or processing intermediates, but also as a white powder sweetener with an elegant sweetness, as well as various foods, beverages, cosmetics, pharmaceuticals, etc. It can be used to advantage in compositions.
[0076]
Example A-5
The mutant strain of Sulfolobus acidocardarius ATCC33909 was cultured in a fermenter for about 42 hours according to the method of Example A-1. After the culture, the cells were concentrated using an SF membrane, about 5 l of the cell suspension was collected, and the suspension was treated in a minilab to disrupt the cells contained. The treated liquid was centrifuged to obtain about 4.8 l of a centrifuged supernatant. Ammonium sulfate was added to the supernatant to a saturation level of about 0.7, the enzyme was salted out, the precipitate was recovered by centrifugation, and dissolved in 10 mM phosphate buffer (pH 6.5). Then, about 600 ml of an enzyme solution containing a thermostable non-reducing saccharide-forming enzyme (about 15 units / ml) and a thermostable trehalose releasing enzyme (about 12 units / ml) was recovered. Next, hydrophobic column chromatography was performed to recover 5850 units of the heat-resistant non-reducing saccharide-forming enzyme and 3960 units of the heat-resistant trehalose-releasing enzyme. To 1 part by weight of potato starch, 6 parts by weight of water and 0.01 part by weight of α-amylase “Neospitase” manufactured by Nagase Seikagaku Co., Ltd. were added, stirred and mixed, and the pH of this suspension was adjusted to 6.2. Thereafter, the starch is gelatinized and liquefied at 85-90 ° C., and the liquefied liquid is heated at 120 ° C. for 10 minutes to inactivate α-amylase. 5, to which no sucrose was previously dialyzed to remove sucrose, and a UF membrane-concentrated pullulanase "Promozyme 200L" sold by Novo Nordisque Bioindustry Co., Ltd. was prepared in an amount of 500 units per gram of starch and by the above method. The heat-resistant non-reducing saccharide-forming enzyme thus obtained was added in an amount of 1 unit per gram of starch, and the heat-resistant trehalose-releasing enzyme was added in an amount of 1 unit per gram of starch, and reacted for 72 hours. The reaction solution was deactivated at 97 ° C. for 30 minutes to inactivate the enzyme, and then adjusted to 50 ° C. and pH 5.0, and glucoamylase “glucozyme” manufactured by Nagase Seikagaku Corporation was added at 10 units per gram of starch. The reaction was allowed to react for 24 hours and then heated to inactivate the enzyme. This solution was decolorized with activated carbon, desalted with an ion exchange resin, and concentrated to a concentration of about 60% according to a conventional method. The sugar solution contained 79.5% trehalose per solid. Column chromatography was performed according to the method of Example A-2, except that a sodium-type strongly acidic cation exchange resin “C6000” sold by Organo Corporation was used as the ion exchange resin, and a trehalose-rich fraction was collected. The high content liquid contained about 95% trehalose per solid. After concentrating the solution to a concentration of 75%, the solution is taken up in an auxiliary crystallizer, and about 2% of trehalose hydrated crystals are added as seed crystals to stir auxiliary crystals, then taken out in a plastic vat and left at room temperature for 3 days to crystallize and mature. Then, a block was prepared. Next, the block was pulverized with a cutting machine to obtain a trehalose hydrated crystal powder at a yield of about 70% based on the solids of the raw starch. This product has practically no hygroscopicity, is easy to handle, and has various compositions such as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. It can be used to advantage for things.
[0077]
Example A-6
Sulfolobus solfataricus ATCC 35091 was cultured in a fermenter for about 42 hours according to the method of Experiment 1. After the culturing, the SF membrane was concentrated and the cells were disrupted according to the method of Example A-1, and the centrifuged supernatant was subjected to ammonium sulfate precipitation, the salted-out product was dialyzed, and ion exchange column chromatography was performed. After collecting the active fraction, it is concentrated on a UF membrane, and a concentrated enzyme solution containing a thermostable non-reducing saccharide-forming enzyme (26.4 units / ml) and a thermostable trehalose releasing enzyme (57.5 units / ml). About 150 ml was collected. Then, hydrophobic column chromatography was performed to recover 2650 units of the thermostable non-reducing saccharide-forming enzyme and 5950 units of the thermostable trehalose releasing enzyme. Potato starch milk having a concentration of 6% was gelatinized by heating, and then adjusted to pH 4.5 and a temperature of 50 ° C., and isoamylase was added thereto at a ratio of 500 units per gram of starch, and reacted for 20 hours. The reaction solution was adjusted to pH 6.5, autoclaved (120 ° C.) for 10 minutes, and then cooled to 95 ° C., to which 0.1% by weight of Novo α-amylase “Termamyl 60L” was added per gram of starch. And reacted for 15 minutes. The reaction solution was subjected to an autoclave (130 ° C.) for 30 minutes, and then cooled to 65 ° C., and the concentrated solution containing the trehalose-releasing enzyme, 1 unit of the non-reducing saccharide-forming enzyme prepared as described above, per gram of starch. One unit was added per gram of starch and reacted for 72 hours. The reaction solution was kept at 97 ° C. for 30 minutes, adjusted to 50 ° C. and pH 5.0, added with 10 units of glucozyme per gram of starch, allowed to react for 24 hours, and then heated to inactivate the enzyme. This solution was decolorized with activated carbon, desalted with an ion exchange resin, and concentrated to a concentration of about 60% according to a conventional method. The sugar solution contained 80.9% trehalose per solid. After concentrating this solution to a concentration of about 84%, it is taken in an auxiliary crystallizer, and about 2% of trehalose hydrated crystal is added as a seed crystal to stir the auxiliary crystal, then taken out in a plastic vat and left at room temperature for 3 days to crystallize. The block was prepared by aging. Next, this block was pulverized with a cutting machine to obtain a trehalose hydrated crystal powder at a yield of about 90% per solid based on the raw starch. This product has practically no hygroscopicity, is easy to handle, and has various compositions such as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. It can be used to advantage for things.
[0078]
Example B-1 Sweetener
To 1 part by weight of the trehalose hydrated crystal powder obtained by the method of Example A-3, 0.01 part by weight of α-glycosyl stevioside “αG suite” sold by Toyo Seika Co., Ltd. and L-aspartyl-L-phenylalanine methyl manufactured by Ajinomoto Co., Ltd. 0.01 part by weight of the ester "aspartame" was uniformly mixed and subjected to a granulating machine to obtain a granular sweetener. The product has an excellent sweetness and a sweetness about 2.5 times that of sucrose, and the calorie per sweetness is reduced to about 1 / 2.5 that of sucrose. This sweetener does not decompose high-intensity sweeteners contained in it and has excellent stability.As a low-calorie sweetener, low-calorie foods for obese and diabetics who have restricted caloric intake It is suitable for sweetening products and the like. The present sweetener is also suitable for sweetening foods and drinks that suppress tooth decay due to little generation of insoluble glucan.
[0079]
[Example B-2 Hard candy]
To 100 parts by weight of a 55% strength sucrose solution, 30 parts by weight of the trehalose-containing syrup obtained by the method of Example A-1 were mixed by heating, and then concentrated under heating under reduced pressure until the water content became less than 2%. A part by weight and an appropriate amount of a lemon flavor and a colorant were mixed and molded according to a conventional method to obtain a product. This product is a high-quality hard candy that is crisp, tastes good, and does not cause crystallization of sucrose.
[0080]
[Example B-3 Chewing gum]
3 parts by weight of the gum base was heated and melted to a degree that it was softened, 4 parts by weight of sucrose and 3 parts by weight of the trehalose hydrated crystal powder obtained by the method of Example A-3 were added, and an appropriate amount of a flavor and a coloring agent were further added. The mixture was mixed, kneaded with a roll, molded and packaged according to a conventional method to obtain a product. This product is a chewing gum with good texture and flavor.
[0081]
Example B-4 Sweetened Condensed Milk
In 100 parts by weight of raw milk, 3 parts by weight of the trehalose-containing syrup and 1 part by weight of sucrose obtained by the method of Example A-1 were dissolved, sterilized by heating with a plate heater, then concentrated to a concentration of 70%, and canned under aseptic conditions. And got the product. This product has a mild sweetness and good flavor, and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, black tea and the like.
[0082]
[Example B-5 Lactic acid bacteria drink]
175 parts by weight of skim milk powder, 130 parts by weight of the trehalose-containing syrup obtained by the method of Example A-1, and Open 50 parts by weight of the lactosucrose-rich powder disclosed in Japanese Unexamined Patent Publication No. 4-287995 is dissolved in 1,150 parts by weight of water, sterilized at 65 ° C. for 30 minutes, cooled to 40 ° C., and added thereto according to a conventional method. Then, 30 parts by weight of a lactic acid bacteria starter was inoculated and cultured at 37 ° C. for 8 hours to obtain a lactic acid bacteria beverage. This product is a lactic acid bacteria beverage having a good flavor. In addition, the product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacterium growth promoting effect.
[0083]
Example B-6 Powdered Juice
50 parts by weight of the trehalose-rich powder obtained by the method of Example A-2, 10 parts by weight of sucrose, 0.65 parts by weight of anhydrous citric acid, 0 parts by weight of malic acid with respect to 33 parts by weight of orange juice powder produced by spray drying. 1 part by weight, 0.1 part by weight of L-ascorbic acid, 0.1 part by weight of sodium citrate, 0.5 part by weight of pullulan, an appropriate amount of powdered fragrance, well mixed and stirred, pulverized to a fine powder to obtain a fluidized bed. Charged into granulator, exhaust air temperature 40 ° C, air volume 150m ³ Then, the trehalose-containing syrup obtained by the method of Example A-1 was sprayed as a binder, granulated for 30 minutes, weighed and packaged to obtain a product. This product is a powdered juice having a fruit juice content of about 30%. This product had no off-flavor or off-flavor and was stable for a long period of time.
[0084]
[Example B-7 Custard cream]
100 parts by weight of corn starch, 100 parts by weight of trehalose-containing syrup obtained by the method of Example A-1, 80 parts by weight of maltose, 20 parts by weight of sucrose, and 1 part by weight of salt are sufficiently mixed, and 280 parts by weight of a chicken egg are added and stirred. Then, slowly add 1,000 parts by weight of boiled milk, further heat the mixture and continue stirring. When the corn starch is completely gelatinized and the whole becomes translucent, stop the fire and cool it. Then, an appropriate amount of vanilla flavor was added, weighed, filled, and packaged to obtain a product. This product has a smooth luster and is mildly sweet and delicious.
[0085]
Example B-8 Uirono Element
90 parts by weight of rice flour, 20 parts by weight of corn starch, 40 parts by weight of sucrose, 80 parts by weight of trehalose hydrated crystal powder obtained by the method of Example A-3, and 4 parts by weight of pullulan were uniformly mixed to produce uronomoto. . Uiro-no-moto, an appropriate amount of matcha and water were kneaded, and this was put in a container and steamed for 60 minutes to produce matcha uiro. This product has good shine, mouthfeel and good flavor. In addition, aging of the starch is suppressed, and the shelf life is good.
[0086]
[Example B-9]
Water was added to 10 parts by weight of the raw material Azuki according to a conventional method, and the mixture was boiled, astringently cut out, and air-extracted to remove water-soluble impurities, thereby obtaining about 21 parts by weight of Azuki tsubuan. To this raw bean paste, 14 parts by weight of sucrose, 5 parts by weight of the trehalose-containing syrup obtained by the method of Example A-1 and 4 parts by weight of water are added and boiled. And about 35 parts by weight of the product was obtained. This product has no color burning, has a good texture, and has a good flavor, and is suitable as an ingredient for anpan bread, steamed bun, dumpling, monaka, frozen dessert and the like.
[0087]
Example B-10 Bread
100 parts by weight of flour, 2 parts by weight of yeast, 5 parts by weight of sugar, 1 part by weight of the trehalose-containing powder obtained by the method of Example A-2, and 0.1 part by weight of an inorganic food are kneaded with water according to a conventional method. The seeds were fermented at 26 ° C. for 2 hours, then aged for 30 minutes and baked. This product is a high-quality bread having good hue, good elasticity, moderate elasticity and mild sweetness.
[0088]
Example B-11 Ham
To 1000 parts by weight of pork leg, 15 parts by weight of salt and 3 parts by weight of potassium nitrate are evenly rubbed, and piled up in a cold room all day and night. This was immersed in a cold room for 7 days in a salting solution comprising 500 parts by weight of water, 100 parts by weight of sodium chloride, 3 parts by weight of potassium nitrate, 40 parts by weight of trehalose hydrated crystal powder obtained by the method of Example A-6 and spices, According to a conventional method, the product was washed with cold water, wound with a string, smoked, cooked, and cooled and packaged to obtain a product. This product is a high quality ham with good color and good flavor.
[0089]
Example B-12 Powder Peptide
2 parts by weight of the trehalose hydrate crystal powder obtained by the method of Example A-6 were mixed with 1 part by weight of a 40% soybean peptide solution for food "Hinute S" manufactured by Fuji Oil Co., Ltd., and put in a plastic vat. It was dried under reduced pressure at 50 ° C. and pulverized to obtain a powdered peptide. This product has a good flavor and can be advantageously used not only as a confectionery material such as premix and frozen dessert, but also as a baby food such as an oral liquid food and a tube food liquid, a therapeutic nutrient and the like.
[0090]
[Example B-13 Powder miso]
1 part by weight of red miso was mixed with 3 parts by weight of anhydrous crystalline trehalose powder obtained by the method of Example A-4, poured into a metal plate provided with a large number of hemispherical concave portions, and allowed to stand at room temperature overnight. It was solidified and cast to obtain about 4 grams of solid miso per piece, which was crushed to obtain powdered miso. This product can be advantageously used as a seasoning for instant noodles, instant soups, etc. The solid miso can be used not only as a solid seasoning but also as a miso confection.
[0091]
[Example B-14 Powdered egg yolk]
The yolk prepared from the raw egg is sterilized at 60 to 64 ° C. with a plate-type heat sterilizer, and 4 parts by weight of the anhydrous crystalline trehalose powder obtained by the method of Example A-4 is used for 1 part by weight of the obtained liquid yolk. After mixing at a ratio, the mixture was transferred to a vat and left overnight to be converted into trehalose hydrated crystals to prepare a block. The block was pulverized by a cutting machine to obtain powdered egg yolk. The product can be advantageously used not only as a confectionary material such as a premix, a frozen dessert, an emulsifier, but also as a baby food such as an oral liquid food and a tube food, a therapeutic nutrient, and the like. In addition, it can also be advantageously used as a beautifying agent, a hair restorer and the like.
[0092]
Example B-15 Cosmetic Cream
2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, 2 parts by weight of trehalose-rich powder obtained by the method of Example A-2, 1 part by weight of α-glycosyl rutin, liquid paraffin 1 part by weight, 10 parts by weight of glycerin trioctanoate and an appropriate amount of a preservative were dissolved by heating according to a conventional method, and 2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water were added thereto. The mixture was emulsified with a homogenizer, and an appropriate amount of a flavor was further added, followed by stirring and mixing to produce a cream. The product has antioxidant properties, high stability, and can be advantageously used as a high-quality sunscreen, skin-beautifying agent, fair-skinning agent, and the like.
[0093]
Example B-16 Powdered Ginseng Extract
After kneading 1.5 parts by weight of anhydrous crystalline trehalose powder obtained by the method of Example A-4 into 0.5 part by weight of ginseng extract, transfer the mixture to a vat, and leave it for 2 days to convert to trehalose hydrated crystals to form a block. Prepared. The block was pulverized by a cutting machine and classified to obtain a powdered ginseng extract. This product was applied to an appropriate amount of vitamin B1 and vitamin B2 powder in a granulating machine to obtain a vitamin-containing granular ginseng extract. This product can be advantageously used as a fatigue recovery agent, tonic, tonic, etc. It can also be used as a hair restorer.
[0094]
Example B-17 Solid preparation
A human natural interferon-α sample (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) is applied to an immobilized anti-human interferon-α antibody column according to a conventional method to adsorb human natural interferon-α contained in the sample. The bovine serum albumin, which is a stabilizer, was removed by passing it through, and then the pH was changed to obtain a physiological solution containing 5% of a trehalose-rich powder obtained by the method of Example A-2 using human natural interferon-α. Elution was carried out using saline. This liquid is subjected to precision filtration, added to about 20 times the amount of anhydrous crystalline maltose powder “Fine Tose” sold by Hayashibara Shoji Co., Ltd., dehydrated and pulverized, and then tableted with a tableting machine to give 1 tablet (about 200 mg). A tablet containing about 150 units of human natural interferon-α per unit was obtained. The product is orally administered as sublingual tablets, for example, about 1 to 10 tablets per day, and can be advantageously used for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., in which the number of patients has rapidly increased in recent years. In this product, maltose acts as a stabilizer together with trehalose, and its activity is well maintained for a long time even at room temperature.
[0095]
Example B-18 Dragee
A 150 mg plain tablet was used as a core, and 40 parts by weight of trehalose hydrated crystalline powder obtained by the method of Example A-3, 2 parts by weight of pullulan (average molecular weight 200,000), 30 parts by weight of water, 25 parts by weight of talc And sugar coating using a undercoating solution consisting of 3 parts by weight of titanium oxide until the tablet weight becomes about 230 mg, and then an overhanging solution consisting of 65 parts by weight of the same trehalose hydrated crystal powder, 1 part by weight of pullulan and 34 parts by weight of water. And coated with a wax solution to obtain a sugar-coated tablet excellent in gloss and appearance. This product has excellent impact resistance and maintains high quality for a long time.
[0096]
[Example B-19 Toothpaste]
Combination
Dicalcium phosphate 45.0%
Pullulan 2.95%
Sodium lauryl sulfate 1.5%
Glycerin 20.0%
Polyoxyethylene sorbitan laurate 0.5%
Preservative 0.05%
Trehalose hydrous crystalline powder obtained by the method in Example B-3 12.0%
Maltitol 5.0%
Water 13.0%
The above-mentioned materials were mixed according to a conventional method to obtain toothpaste. This product has a moderate sweetness and is particularly suitable as a toothpaste for children.
[0097]
[Example B-20 Liquid edible solid preparation]
500 parts by weight of trehalose hydrated crystalline powder produced by the method of Example A-3, 270 parts by weight of powdered egg yolk, 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate Of thiamine, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate, 0.6 part by weight of vitamin E acetate and 0.04 part by weight of nicotinamide, and moisture-proofing 25 g of the mixture at a time. The product was filled in a heat-resistant laminated pouch and heat-sealed to obtain a product. This product is obtained by dissolving one bag in about 150 to 300 ml of water to make a liquid diet, and is used orally or by tube application to the nasal cavity, stomach, intestine, etc., and is advantageous for replenishing energy to living organisms. Available to
[0098]
[Example B-23 Trauma treatment plaster]
To 200 parts by weight of the hydrated trehalose hydrated crystal powder and 300 parts by weight of maltose produced by the method of Example A-3, 50 parts by weight of methanol in which 3 parts by weight of iodine was dissolved were added and mixed, and 200 parts by weight of a 10 w / v% pullulan aqueous solution was further added. Was added and mixed to obtain a wound treatment plaster exhibiting moderate elongation and adhesiveness. Since this product acts not only as a bactericidal action by iodine but also as an energy supplement to cells by trehalose, the healing period is shortened and the wound surface is healed neatly.
[0099]
【The invention's effect】
As is clear from the above, the novel thermostable trehalose-releasing enzyme of the present invention releases trehalose from a non-reducing saccharide having a trehalose structure at a terminal and a degree of polymerization of glucose of 3 or more, and has excellent heat stability. In addition, trehalose is produced in high yield at a high temperature exceeding 55 ° C., which is less contaminated with various bacteria, by acting on the partially decomposed reducing starch together with a heat-resistant non-reducing saccharide-forming enzyme. It is easy to separate and purify the trehalose, and the trehalose and the saccharide containing the trehalose thus obtained are excellent in stability, have good quality and have an elegant sweetness. In addition, it is digested and absorbed by ingestion and becomes a source of calories. Trehalose and saccharides containing it can be advantageously used as sweeteners, taste improvers, quality improvers, stabilizers, excipients and the like in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.
[0100]
Therefore, the establishment of the present invention is based on the fact that trehalose and saccharides containing it, which could not be obtained as easily as previously desired from starch partially decomposed products derived from starch, which is an inexpensive and unlimited resource, can be industrially produced in large quantities. It will open up a whole new way to supply it cheaply, and its impact will be not only in the fields of science such as starch science, enzyme science and biochemistry, but also in industries, especially food, cosmetics and pharmaceuticals. Of course, it extends to the agriculture, fisheries and livestock industries and the chemical industry, and the industrial significance to these industries is immense.
[Brief description of the drawings]
FIG. 1 is a diagram showing elution patterns of a thermostable trehalose releasing enzyme and a non-reducing saccharide-forming enzyme of the present invention from DEAE-Toyopearl.
FIG. 2 is a graph showing the effect of temperature on the enzymatic activity of the thermostable trehalose releasing enzyme of the present invention.
FIG. 3 is a graph showing the effect of pH on the enzyme activity of the thermostable trehalose releasing enzyme of the present invention.
FIG. 4 shows the effect of temperature on the stability of the thermostable trehalose releasing enzyme of the present invention.
FIG. 5 is a graph showing the effect of pH on the stability of the thermostable trehalose releasing enzyme of the present invention.

Claims (20)

Terminus specifically hydrolyze a bond between a trehalose moiety and the remaining glycosyl moiety of glucose polymerization degree having a trehalose structure 3 or more non-reducing saccharides, which can be enzymatic reactions at temperatures above 55 ° C. And a thermostable trehalose releasing enzyme having a molecular weight of about 54,000 to 64,000 daltons obtained by SDS-gel electrophoresis, which can be obtained from a microorganism belonging to the genus Sulfolobus . 2. The thermostable trehalose releasing enzyme according to claim 1, wherein the glycosyl moiety comprises a glucose residue having a degree of polymerization of 1 or more. The heat-resistant trehalose-releasing enzyme according to claim 1 or 2, wherein the heat resistance is stable up to around 85 ° C when kept at pH 7.0 for 60 minutes. A thermostable trehalose-releasing enzyme having the following physicochemical properties and obtainable from a microorganism belonging to the genus Sulfolobus ;
(1) The trehalose is liberated by specifically hydrolyzing the bond between the trehalose moiety of the non-reducing saccharide having a trehalose structure at the working terminal and having a degree of polymerization of 3 or more and the other glycosyl moiety.
(2) Molecular weight: about 54,000 to 64,000 daltons by SDS-gel electrophoresis.
(3) pI of about 5.6 to 6.6 by electrophoresis containing an isoelectric point ampholine.
(4) Optimum temperature: pH 6.0, reaction for 30 minutes, around 75 ° C.
(5) Optimum pH
The reaction is performed at 60 ° C for 30 minutes, and the pH is about 5.5 to 6.0.
(6) Temperature stability Stable up to around 85 ° C by holding at pH 7.0 for 60 minutes.
(7) pH stability A pH of about 4.5 to 9.5 when kept at 25 ° C. for 16 hours. A microorganism belonging to the genus Sulfolobus having the ability to produce a heat-resistant trehalose-releasing enzyme according to any one of claims 1 to 4, wherein the microorganism is cultured in a nutrient medium, and the heat-resistant trehalose-releasing enzyme is collected from the resulting culture. The method for producing a thermostable trehalose releasing enzyme according to any one of claims 1 to 4 . Microorganisms belonging to Sulfolobus is Sulfolobus acidocaldarius (ATCC 33909), Sulfolobus acidocaldarius (ATCC49426), is Sulfolobus solfataricus (ATCC35091), or Sulfolobus solfataricus (ATCC35092), according to claim 5, wherein A method for producing a thermostable trehalose releasing enzyme. The trehalose produced by causing the heat-resistant trehalose-releasing enzyme according to any one of claims 1 to 4 to act on a non-reducing saccharide having a trehalose structure at a terminal and having a degree of polymerization of 3 or more to produce trehalose, or A method for producing trehalose or a saccharide containing the same, comprising collecting a saccharide containing the trehalose. A non-reducing saccharide-forming enzyme having the following physicochemical properties and the heat-resistant trehalose-releasing enzyme according to any one of claims 1 to 4 are allowed to simultaneously act on the reduced starch partially degraded product, or A method for producing trehalose or a saccharide containing the trehalose, wherein the enzyme is allowed to act in this order to produce trehalose, and the generated trehalose or a saccharide containing the trehalose is collected ;
(1) Action
One or two or more non-reducing saccharides having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more are produced from one or more kinds of partially decomposed reductive starch having a glucose polymerization degree of 3 or more.
(2) Molecular weight
Approximately 69000 to 79,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 5.4 to 6.4 by electrophoresis containing ampholine.
(4) Optimal temperature
pH 5.5, 60 minutes reaction, around 75 ° C.
(5) Optimum pH
The pH is around 5.0 to 5.5 at 60 ° C for 60 minutes.
(6) Temperature stability
Stable up to around 85 ° C by holding pH 7.0 for 60 minutes.
(7) pH stability
PH of about 4.0 to 9.5 at 25 ° C for 16 hours. 9. The method for producing trehalose or a saccharide containing the same according to claim 8 , wherein the partially decomposed reductive starch is a partially degraded reductible starch obtained by partially hydrolyzing starch with an enzyme or an acid. The method for producing trehalose or a saccharide containing the same according to claim 9 , wherein the enzyme is an amylase and / or a debranching enzyme. The trehalose according to any one of claims 8 to 10 , wherein the partially decomposed reducing starch is one or more kinds of partially decomposed reducing starch, a liquefied starch, a dextrin, or a maltooligosaccharide having a glucose polymerization degree of 3 or more. Or a method for producing a saccharide containing the same. The method for producing trehalose or a saccharide containing the trehalose according to any one of claims 7 to 11 , further comprising allowing glucoamylase to act. Trehalose or trehalose according to any one of claims 7 to 12 , characterized in that column chromatography using a salt type strongly acidic cation exchange resin is used for collecting a saccharide containing the same. Production method of carbohydrate containing. The method for producing trehalose or a saccharide containing the same according to any one of claims 7 to 13 , wherein the trehalose is a hydrous crystal or an anhydrous crystal. A method for producing food or drink, characterized by including trehalose obtained by the method for producing trehalose according to any one of claims 7 to 14 or a saccharide containing the trehalose. A method for producing cosmetics, characterized by including trehalose obtained by the method for producing trehalose according to any one of claims 7 to 14 , or saccharide containing the trehalose. A method for producing a medicament, characterized by including trehalose obtained by the method for producing trehalose according to any one of claims 7 to 14 or a saccharide containing the same, or a saccharide containing the same. An enzyme solution comprising the thermostable trehalose releasing enzyme according to any one of claims 1 to 4 , and a non-reducing saccharide-forming enzyme having the following physicochemical properties :
(1) Action
One or two or more non-reducing saccharides having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more are produced from one or more kinds of partially decomposed reductive starch having a glucose polymerization degree of 3 or more.
(2) Molecular weight
Approximately 69000 to 79,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 5.4 to 6.4 by electrophoresis containing ampholine.
(4) Optimal temperature
pH 5.5, 60 minutes reaction, around 75 ° C.
(5) Optimum pH
The pH is around 5.0 to 5.5 at 60 ° C for 60 minutes.
(6) Temperature stability
Stable up to around 85 ° C by holding pH 7.0 for 60 minutes.
(7) pH stability
PH of about 4.0 to 9.5 at 25 ° C for 16 hours. A microorganism belonging to the genus Sulfolobus having the ability to produce the thermostable trehalose-releasing enzyme according to any one of claims 1 to 4 and the ability to produce a non-reducing saccharide-forming enzyme having the following physicochemical properties in a nutrient medium. Culturing to produce the heat-resistant trehalose-releasing enzyme and the non-reducing saccharide-forming enzyme, and collecting the heat-resistant trehalose-releasing enzyme and the non-reducing saccharide-forming enzyme from the obtained culture. A method for producing a thermostable trehalose releasing enzyme and a non-reducing saccharide-forming enzyme ;
(1) Action
One or two or more non-reducing saccharides having a trehalose structure at the terminal and having a glucose polymerization degree of 3 or more are produced from one or more kinds of partially decomposed reductive starch having a glucose polymerization degree of 3 or more.
(2) Molecular weight
Approximately 69000 to 79,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 5.4 to 6.4 by electrophoresis containing ampholine.
(4) Optimal temperature
pH 5.5, 60 minutes reaction, around 75 ° C.
(5) Optimum pH
The pH is around 5.0 to 5.5 at 60 ° C for 60 minutes.
(6) Temperature stability
Stable up to around 85 ° C by holding pH 7.0 for 60 minutes.
(7) pH stability
PH of about 4.0 to 9.5 at 25 ° C for 16 hours. Microorganisms belonging to Sulfolobus is Sulfolobus acidocaldarius (ATCC 33909), Sulfolobus acidocaldarius (ATCC49426), is Sulfolobus solfataricus (ATCC35091), or Sulfolobus solfataricus (ATCC35092), according to claim 19, wherein For producing a thermostable trehalose-releasing enzyme and a non-reducing saccharide-forming enzyme.

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